Tissue-protective cytokines for the protection, restoration and enhancement of responsive cells, tissues and organs

ABSTRACT

Methods and compositions are provided for protecting or enhancing a responsive cell, tissue, organ or body part function or viability in vivo, in situ or ex vivo in mammals, including human beings, by systemic or local administration of a tissue protective cytokine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/188,905, filed Jul. 3, 2002, now abandoned, which is acontinuation-in-part, under 35 U.S.C. §111(a), of PCT Patent ApplicationPCT/US01/49479 entitled Protection, Restoration, and Enhancement ofErythropoietin Responsive Cells, Tissues and Organs, filed on Dec. 28,2001, which is incorporated herein by reference in its entirety, andclaims priority under 35 U.S.C. §119(e) (1) to provisional applicationSer. No. 60/259,245 filed on Dec. 29, 2000. Additionally, U.S. patentapplication Ser. No. 10/188,905 is a continuation in part of U.S. patentapplication Ser. No. 09/753,132 entitled Protection and Enhancement ofErythropoietin-Responsive Cells, Tissues, and Organs, filed on Dec. 29,2000, which issued as U.S. Pat. No. 6,531,121 on Mar. 11, 2003, theentire contents of each of which is incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

For many years, the only clear physiological role of erythropoietin hadbeen its control of the production of red blood cells. Recently, severallines of evidence suggest that erythropoietin, as a member of thecytokine superfamily, performs other important physiologic functionswhich are mediated through interaction with the erythropoietin receptor(erythropoietin-R). These actions include mitogenesis, modulation ofcalcium influx into smooth muscle cells and neural cells, production oferythrocytes, hyperactivation of platelets, production of thrombocytes,and effects on intermediary metabolism. It is believed thaterythropoietin provides compensatory responses that serve to improvehypoxic cellular microenvironment as well as modulate programmed celldeath caused by metabolic stress. Although studies have established thaterythropoietin injected intracranially protects neurons against hypoxicneuronal injury, intracranial administration is an impractical andunacceptable route of administration for therapeutic use, particularlyfor normal individuals. Furthermore, previous studies of anemic patientsgiven erythropoietin have concluded that peripherally-administerederythropoietin is not transported into the brain (Marti et al., 1997,Kidney Int. 51:416-8; Juul et al., 1999, Pediatr. Res. 46:543-547; Buemiet al., 2000, Nephrol. Dial. Transplant. 15:422-433).

Various modified forms of erythropoietin have been described withactivities directed towards improving the erythropoietic activity of themolecule, such as those with altered amino acids at the carboxy terminusdescribed in U.S. Pat. No. 5,457,089 and in U.S. Pat. No. 4,835,260;erythropoietin isoforms with various numbers of sialic acid residues permolecule, such as described in U.S. Pat. No. 5,856,298; polypeptidesdescribed in U.S. Pat. No. 4,703,008; agonists described in U.S. Pat.No. 5,767,078; peptides which bind to the erythropoietin receptor asdescribed in U.S. Pat. Nos. 5,773,569 and 5,830,851; and small-moleculemimetics as described in U.S. Pat. No. 5,835,382.

The present invention relates to tissue protective cytokines generatedby the chemical modification of erythropoietin and their uses forprotecting, maintaining, enhancing, or restoringerythropoietin-responsive cells and associated cells, tissues and organsin situ as well as ex vivo, and to delivery of a tissue protectivecytokine across an endothelial cell barrier for the purpose ofprotecting and enhancing erythropoietin-responsive cells and associatedcells, tissues and organs distal to the vasculature, or to carryassociated molecules across an endothelial cell barrier.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to the use of tissueprotective cytokines, chemically-modified erythropoietins that lack oneor more aspects of erythropoietin's affect on the bone marrow, for thepreparation of pharmaceutical compositions for protecting, maintaining,enhancing, or restoring the function or viability of responsivemammalian cells and their associated cells, tissues and organs. In oneparticular aspect, the responsive mammalian cells and their associatedcells, tissues or organs are distal to the vasculature by virtue of atight endothelial cell barrier. In another particular aspect, the cells,tissues, organs or other bodily parts are isolated from a mammalianbody, such as those intended for transplant or reattachment. By way ofnon-limiting examples, the responsive cell or tissue may be neuronal,retinal, muscle, heart, lung, liver, kidney, small intestine, adrenalcortex, adrenal medulla, capillary endothelial, testes, ovary, pancreas,bone, skin or endometrial cells or tissue. Further, non-limitingexamples of responsive cells include photoreceptor (rods and cones),ganglion, bipolar, horizontal, amacrine, Mueller, myocardium, pacemaker, sinoatrial node, sinoatrial node, sinus node, junction tissue,atrioventricular node, bundle of His, hepatocytes, stellate, Kupffer,mesangial, renal epithelial, tubular interstitial, goblet, intestinalgland (crypts), enteral, endocrine, glomerulosa, fasciculate,reticularis, chromaffin, pericyte, Leydig, Sertoli, sperm, Graffianfollicle, primordial follicle, islets of Langerhans, α-cells, β-cells,γ-cells, F-cells, osteoprogenitor, osteoclasts, osteoblasts, endometrialstroma, endometrial, stem and endothelial cells. These examples ofresponsive cells are merely illustrative. In one aspect, the responsivecell or its associated cells, tissues, or organs are not excitablecells, tissues, or organs, or do not predominantly comprise excitablecells or tissues. In a particular embodiment, the mammalian cell, tissueor organ for which an aforementioned tissue protective cytokine is usedare those that have expended or will expend a period of time under atleast one condition adverse to the viability of the cell, tissue ororgan. Such conditions include traumatic in-situ hypoxia or metabolicdysfunction, surgically-induced in-situ hypoxia or metabolicdysfunction, or in-situ toxin exposure; the latter may be associatedwith chemotherapy or radiation therapy. In one embodiment, the adverseconditions are a result of cardio-pulmonary bypass (heart-lung machine),as is used for certain surgical procedures.

The tissue protective cytokines herein are useful for the therapeutic orprophylactic treatment of human diseases of the CNS or peripheralnervous system which have primarily neurological or psychiatricsymptoms, as well as ophthalmic diseases, cardiovascular diseases,cardiopulmonary diseases, respiratory diseases, kidney, urinary andreproductive diseases, gastrointestinal diseases and endocrine andmetabolic abnormalities, and inflammation.

The invention is also directed to pharmaceutical compositions comprisingparticular tissue protective cytokines for administration to a mammaliananimal, preferably a human. Such pharmaceutical compositions may beformulated for oral, intranasal, or parenteral administration, or in theform of a perfusate solution for maintaining the viability of cells,tissues or organs ex vivo.

Tissue protective cytokines useful for the aforementioned purposes andpharmaceutical compositions include erythropoietins that have beenaltered by at least one modification as compared to a nativeerythropoietin, and preferably as compared to native humanerythropoietin. The at least one modification may be a modification ofat least one amino acid of the erythropoietin molecule, or amodification of at least one carbohydrate of the erythropoietinmolecule. Of course, tissue protective cytokine molecules useful for thepurposes herein may have a plurality of modifications compared to anative molecule, such as multiple modifications of the amino acidportion of the molecule, multiple modifications of the carbohydrateportion of the molecule, or at least one modification of the amino acidportion of the molecule and at least one modification of thecarbohydrate portion of the molecule. The tissue protective cytokinemolecule retains its ability of protecting, maintaining, enhancing orrestoring the function or viability of responsive mammalian cells, yetone or more properties of the erythropoietin molecule unrelated to theaforementioned, desirable feature may be absent as compared to thenative molecule. In a preferred embodiment, the tissue protectivecytokine lacks erythropoietin's affects on the bone marrow, i.e.,increased hematocrit (erythropoiesis), vasoconstriction (high bloodpressure), increased blood pressure, hyperactivation of platelets,pro-coagulant activities, and increased production of thrombocytes. Morepreferably, the tissue protective cytokines lack erythropoiesis; mostpreferably the tissue protective cytokines are devoid of all oferythropoietin's effects on the bone marrow.

By way of example, the tissue protective cytokine of the invention maybe asialoerythropoietin. In another example, the tissue protectivecytokine of the invention may be erythropoietin or asialoerythropoietinthat has been reacted with one or more reagents that modify one or moreamino groups of amino acid residues of native erythropoietin orasialoerythropoietin. In a preferred embodiment, the tissue protectivecytokine is nonerythropoietic.

In one embodiment, the tissue protective cytokine is an erythropoietinthat has no sialic acid moieties. In a preferred embodiment, the tissueprotective cytokine is asialoerythropoietin, and most preferably, humanasialoerythropoietin. In another embodiment, the tissue protectivecytokine has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 sialic acidmoieties. Such partially desialylated erythropoietins are referred toherein as hyposialoerythropoietins. They may be prepared by chemical orenzymatic modification of native erythropoietin, or may be obtained byexpression in a system which either does not sialylate the molecule atall or only partially sialylates the erythropoietin. Theasialoerythropoietin and hyposialoerythropoietin of the invention areembraced regardless of the means by which the molecules are prepared.

In another preferred embodiment, the tissue protective cytokinecomprises at least one or more modified lysine residues or amodification of the N-terminal amino group of the erythropoietinmolecule, such modifications as those resulting from reaction of thelysine epsilon amino group or the N-terminal amino group with anamino-group-modifying agent or agents. The modified lysine residue ormodified N-terminal amino group further may be chemically reduced. Inone preferred embodiment, an erythropoietin is biotinylated,carbamylated, succinylated or acetylated at one or more lysine groups orat the N-terminus. In another preferred embodiment, the lysine isreacted with an aldehyde or reducing sugar to form an imine, whichoptionally is then stabilized by chemical reduction such as by usingsodium cyanoborohydride to form an N-alkylated lysine residue such asglucitolyl lysine, or which in the case of reducing sugars may bestabilized by Amadori or Heyns rearrangement to form an alpha-deoxyalpha-amino sugar such as alpha-deoxy-alpha-fructosyllysine. In anotherpreferred embodiment, the lysine or N-terminal amino group iscarbamylated (carbamoylated), such as by virtue of reaction with cyanateion, alkyl-carbamylated, aryl-carbamylated, or aryl-thiocarbamylatedwith an alkyl-isocyanate, aryl-isocyanate, or aryl-isothiocyanate,respectively, or it may be acylated by a reactive alkylcarboxylic orarylcarboxylic acid derivative, such as by reaction with aceticanhydride, succinic anhydride or phthalic anhydride. At least one lysinegroup or the N-terminal amino group may also be trinitrophenyl modifiedby reaction with a trinitrobenzenesulfonic acid, or preferably with oneof its salts. In another embodiment, lysine residues may be modified byreaction with a glyoxal, such as reaction with glyoxal, methylglyoxal or3-deoxyglucosone to form the corresponding alpha-carboxyalkylderivatives.

In another embodiment, a tissue protective cytokine can be generated bymodifying at least one tyrosine residue of erythropoietin by using anelectrophilic reagent, such as but not limited to modification bynitration or iodination, to modify an aromatic ring position.

As noted above, a tissue protective agent useful for the purposes hereinmay have at least one of the aforementioned modifications, but may havemore than one of the above modifications. By way of example of tissueprotective cytokines with one modification to the amino acid portion ofthe molecule and optional modification to the carbohydrate portion ofthe molecule, a tissue protective cytokine is carbamylerythropoietin,carbamylasialoerythropoietin, carbamylhyposialoerythropoietin,acetylerythropoietin, acetylasialoerythropoietin,acetylhypoasialoerythropoietin, succinylerythropoietin,succinylasialoerythropoietin, succinylhyposialoerythropoietin,biotinylerythropoietin, biotinylasialoerythropoietin,biotinylhypsialoerythropoietin, iodoerythropoietin,iodoasialoerythropoietin, iodohyposialoerythropoietin,N-epsilon-carboxymethylerythropoietin,N-epsilon-carboxymethylerythropoietin,N-epsilon-carboxymethylhyposialoerythropoietin, andglucitolylerythropoietin, glucitolylasialoerythropoietin,glucitolylasialohypoerythropoietin. These compounds are merely exemplaryof the modified erythropoietins of the invention. The foregoing trivialnames are merely representative of the modifications of the nativeerythropoietin molecule, and as hereinbefore described, the modificationof the amino group may be on one or more epsilon amino groups of lysineresidues, or the N-terminal amino group, or, in the instance of nitro-or iodo-modified erythropoietins, of one or more tyrosine residues. Anycombination of the foregoing is embraced herein. The present inventionalso embraces compositions, including pharmaceutical compositions,comprising one or more of the aforementioned tissue protective cytokinesAny of such compositions may also include native erythropoietin.

In another aspect of the invention, a method is provided for theprotecting, maintaining, enhancing or restoring the function orviability of responsive mammalian cells and their associated cells,tissues and organs, by administering an effective amount of any one ormore of the aforementioned tissue protective cytokines. In oneparticular aspect of the method, the responsive mammalian cells andtheir associated cells, tissues or organs are distal to the vasculatureby virtue of a tight endothelial cell barrier. In another particularaspect, the cells, tissues, organs or other bodily parts are isolatedfrom a mammalian body, such as those intended for transplant orreattachment. By way of non-limiting examples, the responsive cell ortissue may be neuronal, retinal, muscle, heart, lung, liver, kidney,small intestine, adrenal cortex, adrenal medulla, capillary endothelial,testes, ovary, pancreas, skin, bones, or endometrial cells or tissue.These examples of responsive cells are merely illustrative. In aparticular embodiment, the responsive cell or its associated cells,tissues, or organs are not excitable cells, tissues, or organs, or donot predominantly comprise excitable cells or tissues. In anotherparticular embodiment, the mammalian cell, tissue or organ for which anaforementioned tissue protective cytokine may be administered are thosethat have expended or will expend a period of time under at least onecondition adverse to the viability of the cell, tissue or organ. Suchconditions may include traumatic in-situ hypoxia or metabolicdysfunction, surgically-induced in-situ hypoxia or metabolicdysfunction, or in-situ toxin exposure; the latter may be associatedwith chemotherapy or radiation therapy. In one embodiment, the inventionprotects against the adverse conditions resulting from cardio-pulmonarybypass.

In another aspect of the invention, any of the foregoing tissueprotective cytokines can be used in the preparation of a pharmaceuticalcomposition for ex-vivo treatment of cells, tissues and organs for thepurpose of protecting, maintaining, enhancing, or restoring the functionor viability of responsive mammalian cells and their associated cells,tissues and organs. Such ex-vivo treatment is useful, for example, forthe preservation of cells, tissues or organs for transplant, whetherautotransplant or xenotransplant. The cells, tissue or organ may bebathed in a solution comprising tissue protective cytokines, or theperfusate instilled into the organ through the vasculature or othermeans, to maintain cellular functioning during the period wherein thecells, tissue or organ is not integrated with the vasculature of thedonor or recipient. Administration of the perfusate may be made to adonor prior to organ harvesting, as well as to the harvested organ andto the recipient. Moreover, the aforementioned use of any tissueprotective cytokine is useful whenever a cell, tissue or organ isisolated from the vasculature of the individual and thus essentiallyexisting ex vivo for a period of time, the term isolated referring torestricting or clamping the vasculature of or to the cell, tissue, organor bodily part, such as may be performed during surgery, including, inparticular, cardio-pulmonary bypass surgery; bypassing the vasculatureof the cell, tissue, organ or bodily part; removing the cell, tissue,organ or bodily part from the mammalian body, such may be done inadvance of xenotransplantation or prior to and duringautotransplantation; or traumatic amputation of a cell, tissue, organ orbodily part. Thus, this aspect of the invention pertains both to theperfusion with a tissue protective cytokine in situ and ex vivo. Exvivo, the erythropoietin may be provided in a cell, tissue or organpreservation solution. For either aspect, the exposing may be by way ofcontinuous perfusion, pulsatile perfusion, infusion, bathing, injection,or catheterization.

In yet a further aspect, the invention is directed to a method forprotecting, maintaining, enhancing, or restoring the viability of amammalian cell, tissue, organ or bodily part which includes a responsivecell or tissue, in which the cell, tissue, organ or bodily part isisolated from the mammalian body. The method includes at least exposingthe isolated mammalian cell, tissue, organ or bodily part to an amountof a tissue protective cytokine as mentioned above for a duration whichis effective to protect, maintain, enhance, or restore the aforesaidviability. In non-limiting examples, isolated refers to restricting orclamping the vasculature of or to the cell, tissue, organ or bodilypart, such as may be performed during surgery, in particular,cardio-pulmonary bypass surgery; bypassing the vasculature of the cell,tissue, organ or bodily part; removing the cell, tissue, organ or bodilypart from the mammalian body, such may be done in advance ofxenotransplantation or prior to and during autotransplantation; ortraumatic amputation of a cell, tissue, organ or bodily part. Thus, thisaspect of the invention pertains both to the perfusion with a tissueprotective cytokine in situ and ex vivo. Ex vivo, the tissue protectivecytokine may be provided in a cell, tissue or organ preservationsolution. For either aspect, the exposing may be by way of continuousperfusion, pulsatile perfusion, infusion, bathing, injection, orcatheterization.

By way of non-limiting examples, the aforementioned ex-vivo responsivecell or tissue may be or comprise neuronal, retinal, muscle, heart,lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla,capillary endothelial, testes, ovary, pancreas, skin, bone, bone marrow,umbilical chord blood or endometrial cells or tissue. These examples ofresponsive cells are merely illustrative.

All of the foregoing methods and uses are preferably applicable to humanbeings, but are useful as well for any mammal, such as but not limitedto companion animals, domesticated animals, livestock and zoo animals.Routes of administration of the aforementioned pharmaceuticalcompositions include oral, intravenous, intranasal, topical,intraluminal, inhalation or parenteral administration, the latterincluding intravenous, intraarterial, subcutaneous, intramuscular,intraperitoneal, submucosal or intradermal. For ex-vivo use, a perfusateor bath solution is preferred. This includes perfusing an isolatedportion of the vasculature in situ.

In yet another aspect of the invention, any of the aforementioned tissueprotective cytokines are useful in preparing a pharmaceuticalcomposition for restoring a dysfunctional cell, tissue or organ whenadministered after the onset of the disease or condition responsible forthe dysfunction. By way of non-limiting example, administration of apharmaceutical composition comprising tissue protective cytokinesrestores cognitive function in animals previously having brain trauma,even when administered long after (e.g., three days, five days, a week,a month, or longer) the trauma has subsided.

In yet another embodiment, the invention provides methods for the use ofthe aforementioned tissue protective cytokine for restoring adysfunctional cell, tissue or organ when administered after the onset ofthe disease or condition responsible for the dysfunction. By way ofnon-limiting example, methods for administration of a pharmaceuticalcomposition comprising a tissue protective cytokine restores cognitivefunction in animals previously having brain trauma, even whenadministered long after (e.g., three days, five days, a week, a month,or longer) the trauma has subsided. Tissue protective cytokines usefulfor such methods include any of the particular aforementioned modifiederythropoietins

In still yet a further aspect of the present invention, methods areprovided for facilitating the transcytosis of a molecule across anendothelial cell barrier in a mammal by administration of a compositionof a molecule in association with a tissue protective cytokine asdescribed hereinabove. The association between the molecule to betransported and the tissue protective cytokine may be, for example, alabile covalent bond, a stable covalent bond, or a noncovalentassociation with a binding site for the molecule. Endothelial cellbarriers may be the blood-brain barrier, the blood-eye barrier, theblood-testes barrier, the blood-ovary barrier and the blood-placentabarrier. Suitable molecules for transport by the method of the presentinvention include hormones such as growth hormone, nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophicfactor (CNTF), basic fibroblast growth factor (bFGF), transforminggrowth factor β1 (TGFβ1), transforming growth factor β2 (TGFβ2),transforming growth factor β3 (TGFβ3), interleukin 1, interleukin 2,interleukin 3, and interleukin 6, AZT, antibodies against tumor necrosisfactor, antiviral, and immunosuppressive agents such as cyclosporin.Additionally, dyes or markers may be attached to erythropoietin or oneof the tissue protective cytokines of the present invention in order tovisualize cells, tissues, or organs within the brain and other barrieredorgans for diagnostic purposes.

It is a further aspect of the present invention to provide a compositionfor facilitating the transcytosis of a molecule across an endothelialcell barrier in a mammal, the composition comprising the molecule inassociation with a tissue protective cytokine as mentioned hereinabove.The association may be, for example, a labile covalent bond, a stablecovalent bond, or a noncovalent association with a binding site for themolecule. Endothelial cell barriers may be the blood-brain barrier, theblood-eye barrier, the blood-testes barrier, the blood-ovary barrier andthe blood-placenta barrier. Suitable molecules for transport by themethod of the present invention include hormones such as growth hormone,nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),ciliary neurotrophic factor (CNTF), basic fibroblast growth factor(bFGF), transforming growth factor β1 (TGFβ1), transforming growthfactor β2 (TGFβ2), transforming growth factor β3 (TGFβ3), interleukin 1,interleukin 2, interleukin 3, and interleukin 6, AZT, antibodies againsttumor necrosis factor, antiviral, and immunosuppressive agents such ascyclosporin. Additionally, dyes or markers may be attached toerythropoietin or one of the tissue protective cytokines of the presentinvention in order to visualize cells, tissues, or organs within thebrain and other barriered organs for diagnostic purposes.

In a still further aspect of the present invention, any of theaforementioned tissue protective cytokines is useful in preparing apharmaceutical composition for facilitating the transcytosis of amolecule across an endothelial cell barrier in a mammal. The associationmay be, for example, a labile covalent bond, a stable covalent bond, ora noncovalent association with a binding site for the molecule.Endothelial cell barriers may be the blood-brain barrier, the blood-eyebarrier, the blood-testes barrier, the blood-ovary barrier and theblood-placenta barrier. Suitable molecules for transport by the methodof the present invention include hormones, such as growth hormone,antibiotics, antivirals, dyes, markers, and anti-cancer agents, to namebut a few non-limiting examples.

These and other aspects of the present invention will be betterappreciated by reference to the following Figures and DetailedDescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the distribution of erythropoietin receptor in normal humanbrain, in thin sections stained with an anti-erythropoietin antibody.

FIG. 2 is a higher power magnification of the image in FIG. 1.

FIG. 3 shows, using gold-labeled secondary antibodies, theultramicroscopic distribution of erythropoietin receptors.

FIG. 4, prepared similarly to FIG. 3, shows high density erythropoietinreceptors at the luminal and anti-luminal surfaces of human braincapillaries.

FIG. 5 compares the in-vitro efficacy of erythropoietin andasialoerythropoietin on the viability of serum-starved P19 cells.

FIG. 6 is another experiment which compares the in-vitro efficacy oferythropoietin and asialoerythropoietin on the viability ofserum-starved P19 cells.

FIG. 7 shows protection of erythropoietin and asialoerythropoietin in arat focal cerebral ischemia model.

FIG. 8 shows a dose response comparing the efficacy of humanerythropoietin and human asialoerythropoietin in middle cerebral arteryocclusion in a model of ischemic stroke

FIG. 9 shows the activity of iodinated erythropoietin in the P19 assay.

FIG. 10 shows the effect of biotinylated erythropoietin andasialoerythropoietin in the P19 assay.

FIG. 11 compares the in-vitro efficacy of erythropoietin andphenylglyoxal-modified erythropoietin on the viability of serum-starvedP19 cells.

FIG. 12 shows the effect of tissue protective cytokines in the waterintoxication assay.

FIG. 13 depicts the translocation of parenterally-administerederythropoietin into the cerebrospinal fluid.

FIG. 14 shows the maintenance of the function of a heart prepared fortransplantation by erythropoietin.

FIG. 15 shows the protection of the myocardium from ischemic damage byerythropoietin after temporary vascular occlusion.

FIG. 16 depicts the effects of erythropoietin treatment in a ratglaucoma model.

FIG. 17 shows the extent of preservation of retinal function byerythropoietin in the rat glaucoma model.

FIG. 18 depicts the restoration of cognitive function following braintrauma by administration of erythropoietin starting five days aftertrauma.

FIG. 19 depicts the restoration of cognitive function following braintrauma by administration of erythropoietin starting 30 days aftertrauma.

FIG. 20 depicts the efficacy of human asialoerythropoietin in a kainatemodel of cerebral toxicity.

FIG. 21 depicts the efficacy of tissue protective cytokines in a ratspinal cord injury model.

FIG. 22 shows the efficacy of tissue protective cytokines within arabbit spinal cord injury model.

FIG. 23 shows a coronal section of the brain cortical layer stained byhematoxilyn and eosin.

FIG. 24 shows coronal sections of frontal cortex adjacent to the regionof infarction stained by GFAP antibody.

FIG. 25 shows coronal sections of brain cortical layer stained by OX-42antibody.

FIG. 26 shows coronal sections of brain cortical layer adjacent to theregion of infarction stained by OX-42 antibody.

FIG. 27 shows the efficacy of erythropoietin against inflammation in anEAE model.

FIG. 28 compares the affects of dexamethasone and erythropoietin oninflammation in the EAE model.

FIG. 29 shows that erythropoietin suppresses inflammation associatedwith neuronal death.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the invention provide for the local or systemicprotection or enhancement of cells, tissues and organs within amammalian body, under a wide variety of normal and adverse conditions,or protection of those which are destined for relocation to anothermammalian body. In addition, restoration or regeneration of dysfunctionis also provided. As mentioned above, the ability of an erythropoietinto cross a tight endothelial cell barrier and exert its positive effectson responsive cells (as well as other types of cells) distal to thevasculature offers the potential to prevent as well as treat a widevariety of conditions and diseases which otherwise cause significantcellular and tissue damage in an animal, including human, and moreover,permit success of heretofore unattemptable surgical procedures for whichrisk traditionally outweighed the benefits.

Erythropoietin is a glycoprotein hormone which in humans has a molecularweight of about 34 kDa. The mature protein comprises 165 amino acids,and the glycosyl residues comprise about 40% of the weight of themolecule. Erythropoietin can be obtained commercially, for example,under the trademarks of PROCRIT, available from Ortho Biotech Inc.,Raritan, N.J., and EPOGEN, available from Amgen, Inc., Thousand Oaks,Calif. Furthermore, a variety of host systems may be used for expressionand production of recombinant erythropoietin, including, but not limitedto, bacteria, yeast, insect, plant, and mammalian, including human, cellsystems. For example, recombinant erythropoietin produced in bacteria,which do not glycosylate or sialate the product, could be used toproduce non-glycosylated forms of erythropoietin. Alternatively,recombinant erythropoietin can be produced in other systems that doglycosylate, e.g., plants, including human cells. The forms oferythropoietin useful in the practice of the present invention encompasschemical modifications and/or expression-system-mediated glycosylationmodifications of naturally-occurring, synthetic and recombinant forms ofhuman and other mammalian erythropoietins.

“Responsive cell” refers to a mammalian cell whose function or viabilitymay be maintained, promoted, enhanced, regenerated, or in any other waybenefited, by exposure to an erythropoietin. Non-limiting examples ofsuch cells include neuronal, retinal, muscle, heart, lung, liver,kidney, small intestine, adrenal cortex, adrenal medulla, capillaryendothelial, testes, ovary, pancreas, skin, bone and endometrial cells.In particular, responsive cells include, without limitation, neuronalcells; retinal cells: photoreceptor (rods and cones), ganglion, bipolar,horizontal, amacrine, and Mueller cells; muscle cells; heart cells:myocardium, pace maker, sinoatrial node, sinoatrial node, sinus node,and junction tissue cells (atrioventricular node and bundle of his);lung cells; liver cells: hepatocytes, stellate, and Kupffer cells;kidney cells: mesangial, renal epithelial, and tubular interstitialcells; small intestine cells: goblet, intestinal gland (crypts) andenteral endocrine cells; adrenal cortex cells: glomerulosa, fasciculate,and reticularis cells; adrenal medulla cells: chromaffin cells;capillary cells: pericyte cells; testes cells: Leydig, Sertoli, andsperm cells and their precursors; ovary cells: Graffian follicle andprimordial follicle cells; endometrial cells: endometrial stroma andendometrial cells; pancreas cell: islet of Langerhans, α-cells, β-cells,γ-cells, and F-cells; skin cells; bone cells: osteoprogenitor,osteoclast and osteoblast cells; as well as the stem and endothelialcells present in the above listed organs. Moreover, such responsivecells and the benefits provided thereto by an erythropoietin may beextended to provide protection or enhancement indirectly to other cellsthat are not directly responsive, or of tissues or organs which containsuch non-responsive cells. These other cells, or tissues or organs whichbenefit indirectly from the enhancement of responsive cells present aspart of the cells, tissue or organ as “associated” cells, tissues andorgans. Thus, benefits of an erythropoietin as described herein may beprovided as a result of the presence of a small number or proportion ofresponsive cells in a tissue or organ, for example, excitable orneuronal tissue present in such tissue, or the Leydig cells of thetestis, which makes testosterone. In one aspect, the responsive cell orits associated cells, tissues, or organs are not excitable cells,tissues, or organs, or do not predominantly comprise excitable cells ortissues.

The duration and degree of purposeful adverse conditions induced forultimate benefit, such as high-dose chemotherapy, radiation therapy,prolonged ex-vivo transplant survival, and prolonged periods ofsurgically-induced ischemia, may be carried out by taking advantage ofthe invention herein. However, the invention is not so limited, butincludes as one aspect, methods or compositions wherein the targetresponsive cells are distal to the vasculature by virtue of anendothelial-cell barrier or endothelial tight junctions. In general, theinvention is directed to any responsive cells and associated cells,tissues and organs which may benefit from exposure to erythropoietin.Furthermore, cellular, tissue or organ dysfunction may be restored orregenerated after an acute adverse event (such as trauma) by exposure toerythropoietin.

The invention is directed generally to the use of erythropoietin for thepreparation of pharmaceutical compositions for the aforementionedpurposes in which cellular function is maintained, promoted, enhanced,regenerated, or in any other way benefited. The invention is alsodirected to methods for maintaining, enhancing, promoting, orregenerating cellular function by administering to a mammal an effectiveamount of erythropoietin as described herein. The invention is furtherdirected to methods for maintaining, promoting, enhancing, orregenerating cellular function ex vivo by exposing cells, a tissue ororgan to erythropoietin. The invention is also directed to a perfusatecomposition comprising erythropoietin for use in organ or tissuepreservation.

The various methods of the invention utilize a pharmaceuticalcomposition which at least includes erythropoietin at an effectiveamount for the particular route and duration of exposure to exertpositive effects or benefits on responsive cells within or removed froma mammalian body. Where the target cell, tissues or organs of theintended therapy require erythropoietin to cross an endothelial cellbarrier, the pharmaceutical composition includes erythropoietin at aconcentration which is capable, after crossing the endothelial cellbarrier, of exerting its desirable effects upon the responsive cells.Molecules capable of interacting with the erythropoietin receptor andmodulating the activity of the receptor, herein referred to aserythropoietin or erythropoietin receptor activity modulators, areuseful in the context of the present invention. These molecules may be,for example, naturally-occurring, synthetic, or recombinant forms oferythropoietin molecules, as described above, or other molecules whichmay not necessarily resemble erythropoietin in any manner, except tomodulate erythropoietin responsive cell activity, as described herein.

In addition to the above identified tissue protective attributes,erythropoietin is more commonly associated with its effects on the bonemarrow, i.e., increased hematocrit (erythropoiesis), vasoconstriction(high blood pressure), hyperactivation of platelets, pro-coagulantactivity, and increased production of thrombocytes. However, theseeffects on the bone marrow may pose a risk in the chronic and acuteadministration of erythropoietin to treat the cellular, tissue, or organdysfunctions discussed above. Therefore, the invention is directedgenerally to the use of tissue protective cytokines that consist ofchemically modified erythropoietin, which preferably lack one or more oferythropoietin's effects on the bone marrow. More preferably, the tissueprotective cytokines lack erythropoiesis; most preferably the tissueprotective cytokines are devoid of all of erythropoietin's effects onthe bone marrow. In other embodiments, the tissue protective cytokinelacks any two of the aforesaid effects, or any three of the aforesaideffects.

Furthermore, the tissue protective cytokines desirable for the usesdescribed herein may be generated by guanidination, amidination,carbamylation (carbamoylation), trinitrophenylation, acetylation,succinylation, nitration, or modification of arginine, lysine, tyrosine,tryptophan, or cysteine residues or carboxyl groups, among otherprocedures, such as limited proteolysis, removal of amino groups, and/ormutational substitution of arginine, lysine, tyrosine, tryptophan, orcysteine residues of erythropoietin by molecular biological techniques.Preferably, these chemical modifications affect the four recognizedreceptor regions—VLQRY (SEQ ID NO:1), TKVNFYAW (SEQ ID NO:2), SGIRSLTTL(SEQ ID NO:3), or SNFLRG (SEQ ID NO:4). More preferably, these receptorregions, which are basic in nature, are modified by chemicalmodification of the basic amino acids, arginine and lysine, within theseregions. Additionally, the areas of the molecule surrounding thesereceptor regions may be chemically modified as well to affect thekinetics or receptor binding properties of the molecule. This producestissue protective cytokines which maintain an adequate level ofactivities for specific organs and tissues but not for others, such aserythrocytes (e.g., Satake et al; 1990, Biochim. Biophys. Acta1038:125-9; incorporated herein by reference in its entirety), in whichin vivo biological activity was determined by the polycythemic mousebioassay. One non-limiting example as described hereinbelow is themodification of erythropoietin arginine residues by reaction with aglyoxal such as phenylglyoxal (according to the protocol of Takahashi,1977, J. Biochem. 81:395-402). As will be seen below, such a tissueprotective cytokine molecule fully retains its neurotrophic effect. Suchtissue protective cytokine molecules are fully embraced for the varioususes and compositions described herein.

The activity (in units) of erythropoietin and erythropoietin-likemolecules is traditionally defined based on its effectiveness instimulating red cell production in rodent models (and as derived byinternational standards of erythropoietin). One unit (U) of regularerythropoietin (MW of ˜34,000) is about 8 ng of protein (1 mg protein isapproximately 125,000 U). However, as the effect on erythropoiesis isincidental to the desired activities herein and may not necessarily be adetectable property of certain of the tissue protective cytokines of theinvention, a definition of activity of certain tissue protectivecytokines of the invention based on erythropoietic activity isinappropriate. Thus, as used herein, the activity unit of erythropoietinor the tissue protective cytokines is defined as the amount of proteinrequired to elicit the same activity in neural or other responsivecellular systems as is elicited by WHO international standarderythropoietin in the same system. The skilled artisan will readilydetermine the units of a non-erythropoietic erythropoietin or relatedtissue protective cytokine molecule following the guidance herein.

Further to the above-mentioned tissue protective cytokines, thefollowing discussion expands on the various tissue protective cytokinesof the invention.

A tissue protective cytokine of the invention may consist oferythropoietin having at least no sialic acid moieties, referred to asasialoerythropoietin. Preferably, a tissue protective cytokine of theinvention is human asialoerythropoietin. It may be prepared bydesialylating erythropoietin using a sialidase, such as is described inthe manufacturer's packaging for Sialydase A from ProZyme Inc., SanLeandro, Calif. Typically, PROZYME® GLYCOPRO® sequencing-grade SIALYDASEATM (N-acetylneuraminate glycohydrolase, EC 3.2.1.18) is used to cleaveall non-reducing terminal sialic acid residues from complexcarbohydrates and glycoproteins such as erythropoietin. It will alsocleave branched sialic acids (linked to an internal residue). SialydaseA is isolated from a clone of Arthrobacter ureafaciens.

In a non-limiting example of the foregoing procedure, erythropoietin maybe subjected to desialylation by sialidase (0.025 U/mg EPO) at 37 C for3 h, after which the erythropoietin may be desalted and concentrated.After passing over an ion exchange column using the AKTAPRIME™ system(Amersham Pharmacia Biotech), and elution with selected buffers, thefractions containing only the top two bands identified byimmunoelectrophoresis (migrating at pI ˜8.5 and ˜7.9 on IEF gel) areselected. No significant amount of sialic acid should be detected inthis preparation of the tissue protective cytokine.

In alternative embodiments, the tissue protective cytokine of theinvention may be an erythropoietin having at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or 13 sialic acid residues, by partial desialylationby the aforementioned method. These tissue protective cytokinesresulting form the partial desialylation of erythropoietin may also bereferred to herein as hyposialoerythropoietins, and may be a homogeneouscomposition, with, for example, only 2 sialic acids per erythropoietinmolecule, or may be a heterogeneous mixture of a variety of differentdegrees of sialylation, or, for example, having a low number, such asabout 1 to about 4 sialic acid molecules on average per erythropoietin,or, in another example, a higher number, such as about 10 to about 13sialic acids on average per erythropoietin molecule. Such mixtures mayinclude asialoerythropoietin or erythropoietin.

An erythropoietin for the aforementioned uses may have at least one ormore modified arginine residues. For example, the modifiederythropoietin may comprise an R-glyoxal moiety on the one or morearginine residues, where R may be an aryl, heteroaryl, lower alkyl,lower alkoxy, or cycloalkyl group, or an alpha-deoxyglycitolyl group. Asused herein, the term lower “alkyl” means a straight- or branched-chainsaturated aliphatic hydrocarbon group preferably containing 1-6 carbonatoms. Representative of such groups are methyl, ethyl, isopropyl,isobutyl, butyl, pentyl, hexyl and the like. The term “alkoxy” means alower alkyl group as defined above attached to the remainder of themolecule by oxygen. Examples of alkoxy include methoxy, ethoxy, propoxy,isopropoxy and the like. The term “cycloalkyl” refers to cyclic alkylgroups with from three to up to about 8 carbons, including for examplecyclopropyl, cyclobutyl, cyclohexyl and the like. The term aryl refersto phenyl and naphthyl groups. The term heteroaryl refers toheterocyclic groups containing 4-10 ring members and 1-3 heteroatomsselected from the group consisting of oxygen, nitrogen and sulfur.Examples include but are not limited to isoxazolyl, phenylisoxazolyl,furyl, pyrimidinyl, quinolyl, tetrahydroquinolyl, pyridyl, imidazolyl,pyrrolidinyl, 1,2,4-triazoylyl, thiazolyl, thienyl, and the like. The Rgroup may be substituted, as for example the 2,3,4-trihydroxybutyl groupof 3-deoxyglucosone. Typical examples of R-glyoxal compounds areglyoxal, methylglyoxal, 3-deoxyglucosone, and phenylglyoxal. PreferredR-glyoxal compounds are methylglyoxal or phenylglyoxal. An exemplarymethod for such modification may be found in Werber et al., 1975, Isr.J. Med. Sci. 11(11): 1169-70, using phenylglyoxal.

In a further example, at least one arginine residue may be modified byreaction with a vicinal diketone such as 2,3-butanedione orcyclohexanedione, preferably in ca. 50 millimolar borate buffer at pH8-9. A procedure for the latter modification with 2,3-butanedione may becarried out in accordance with Riordan, 1973, Biochemistry 12(20):3915-3923; and that with cyclohexanone according to Patthy et al., 1975,J. Biol. Chem 250(2): 565-9.

A tissue protective cytokine of the invention may comprise at least oneor more modified lysine residues or a modification of the N-terminalamino group of the erythropoietin molecule, such modifications as thoseresulting from reaction of the lysine residue with anamino-group-modifying agent. For example, erythropoietin oraforementioned asialoerythropoietin or hyposialoerythropoietin, may bemodified by acetylation, carbamylation, succinylation, oxidation andsubsequent carboxymethyllysination, among other methods, to modify aminogroups.

In a non-limiting example, tissue protective cytokine may be generatedby carbamylating an erythropoietin, or a desialylated erythropoietinsuch as asialoerythropoietin, with recrystallized potassium cyanate inborate buffer, after which thorough dialysis is performed.

Likewise, an aforementioned erythropoietin may be succinylated byreaction with succinic anhydride, followed by dialysis to form a tissueprotective cytokine of the present invention.

In yet another embodiment, a tissue protective cytokine may be generatedby reacting erythropoietin with acetic anhydride in phosphate buffer toacetylate the erythropoietin. This reaction may be stopped by dialysisagainst water. The method is described in Satake et al, (1990). Chemicalmodification of erythropoietin: an increase in in-vitro activity byguanidination. Biochimica et Biophysica Acta. 1038: 125-129.

In another embodiment, the tissue protective cytokines areN^(ε)-(carboxymethyl)lysine (CML) adducts from erythropoietin orasialoerythropoietin prepared by reaction with glyoxylic acid andNaBH₃CN in sodium phosphate buffer, followed by dialysis. Akhtar et al.,(1999) Conformational study of N^(ε)-(carboxymethyl)lysine adducts ofrecombinant a-crystallins. Current Eye Research, 18: 270-276.

In another embodiment, a tissue protective cytokine is generated bymodifying the lysine residues of erythropoietin by reaction with glyoxalderivatives, such as reaction with glyoxal, methylglyoxal and3-deoxyglucosone to form alpha-carboxyalkyl derivatives. Examplesinclude reaction with glyoxal to form a carboxymethyllysine residue asin Glomb and Monnier, 1995, J. Biol. Chem. 270(17):10017-26, or withmethylglyoxal to form a (1-carboxyethyl)lysine residue as in Degenhardtet al., 1998, Cell. Mol. Biol. (Noisy-le-grand) 44(7):1139-45. Themodified lysine residue further may be chemically reduced. For example,the erythropoietin may be biotinylated via lysine groups, such as inaccordance with the method described in Example 2, in whichD-biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester was reactedwith erythropoietin, followed by removal of unreacted biotin by gelfiltration on a Centricon 10 column, as described by Wojchowski andCaslake, 1989, Blood 74(3):952-8. In this paper, the authors use threedifferent methods of biotinylating erythropoietin, any of which may beused for the preparation of the tissue protective cytokines for the usesherein. Biotin may be added to (1) the sialic acid moieties (2)carboxylate groups or (3) amino groups.

In another preferred embodiment, the lysine may be reacted with analdehyde or reducing sugar to form an imine, which may be stabilized byreduction as with sodium cyanoborohydride to form an N-alkylated lysineresidue such as glucitolyl lysine, or which in the case of reducingsugars may be stabilized by Amadori or Heyns rearrangement to form analpha-deoxy alpha-amino sugar such as alpha-deoxy-alpha-fructosyllysineresidue in the erythropoietin molecule. As an example, preparation of afructosyllysine-modified protein by incubation with 0.5 M glucose insodium phosphate buffer at pH 7.4 for 60 days is described by Makita etal., 1992, J. Biol. Chem. 267:5133-5138. In another example, the lysinegroup may be carbamylated, such as by virtue of reaction with cyanateion, or alkyl- or aryl-carbamylated or -thiocarbamylated with an alkyl-or aryl-isocyanate or -isothiocyanate, or it may be acylated by areactive alkyl- or arylcarboxylic acid derivative, such as by reactionwith acetic anhydride or succinic anhydride or phthalic anhydride.Exemplary are the modification of lysine groups with4-sulfophenylisothiocyanate or with acetic anhydride, both as describedin Gao et al., 1994, Proc Natl Acad Sci USA 91(25):12027-30. Lysinegroups may also be trinitrophenyl modified by reaction withtrinitrobenzenesulfonic acid or preferably its salts. Such methods aredescribed below in Example 2.

At least one tyrosine residue of an erythropoietin may be modified in anaromatic ring position by an electrophilic reagent, such as by nitrationor iodination to generate a tissue protective cytokine By way ofnon-limiting example, erythropoietin may be reacted withtetranitromethane (Nestler et al., 1985, J. Biol. Chem. 260(12):7316-21;or iodinated as described in Example 3. For example, iodination with NaIand IODO-GEN Pre-Coated Iodination Tube (Pierce, 28601), may be carriedout using erythropoietin or asialoerythropoietin in sodium phosphatebuffer.

At least an aspartic acid or a glutamic acid residue of anerythropoietin may be modified, such as by reaction with a carbodiimidefollowed by reaction with an amine such as but not limited toglycinamide.

In another example, a tryptophan residue of an erythropoietin may bemodified, such as by reaction with n-bromosuccinimide orn-chlorosuccinimide, following methods such as described in Josse etal., Chem Biol Interact 1999 May 14; 119-120.

In yet another example, a tissue protective cytokine may be prepared byremoving at least one amino group of a native erythropoietin, such maybe achieved by reaction with ninhydrin followed by reduction of thesubsequent carbonyl group by reaction with borohydride.

In still a further example, a tissue protective cytokine is providedthat has at least an opening of at least one of the cystine linkages inthe erythropoietin molecule by reaction with a reducing agent such asdithiothreitol, followed by reaction of the subsequent sulfhydryls withiodoacetamide, iodoacetic acid or another electrophile to preventreformation of the disulfide linkages. As noted above, alternatively orin combination, disulfide linkages may be abolished by altering acysteine molecule that participates in the actual cross-link or at leastone other amino acid residue that results in the inability of theerythropoietin to form at least one of the disulfide linkages present inthe native molecule.

A tissue protective cytokine may be prepared by subjecting anerythropoietin to a limited chemical proteolysis that targets specificresidues, for example, to cleave after tryptophan residues. Suchresulting erythropoietin fragments are embraced herein.

As noted above, a tissue protective cytokine useful for the purposesherein may have at least one of the aforementioned modifications, butmay have more than one of the above modifications. By way of example ofa tissue protective cytokine with one modification to the carbohydrateportion of the molecule and one modification to the amino acid portion,a tissue protective cytokine may be asialoerythropoietin and have itslysine residues biotinylated or carbamylated.

Moreover, the chemically modified erythropoietin may be further modifiedby mutating at least one amino acid of the erythropoietin. Suchmutations may include substitutions, deletions, including internaldeletions, additions, including additions yielding fusion proteins, orconservative substitutions of amino acid residues within and/or adjacentto the amino acid sequence, but that result in a “silent” change, inthat the change produces a functionally equivalent erythropoietin.Conservative amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Alternatively, non-conservative amino acid changes, andlarger insertions and deletions may be used to create functionallyaltered erythropoietin. Such mutants can be used to alter erythropoietinproperties in desirable ways. For example, in one embodiment, anerythropoietin useful for the practice of the invention can be alteredin one or more amino acids within the four functional domains oferythropoietin which affect receptor binding: VLQRY (SEQ ID NO:1) and/orTKVNFYAW (SEQ ID NO:2) and/or SGLRSLTTL (SEQ ID NO:3) and/or SNFLRG (SEQID NO:4). In another embodiment, erythropoietins containing mutations inthe surrounding areas of the molecule which affect the kinetics orreceptor-binding properties of the molecule can be used.

These additional modifications may be used to enhance the tissueprotective effect, suppress the bone marrow effect, or alter thephysical properties, such as charge, of the tissue protective cytokine.

The foregoing exemplary methods for preparing tissue protectivecytokines of the invention are merely illustrative and non-limiting, andthese or other methods may be used to prepare the compounds of theinvention. The names hereinabove wherein the method of preparation iscontained within the name, such as “acetylated” or “biotinylated,” areprovided herein merely as a means for understanding how the compound wasmade, yet the present invention is directed to the compounds that areproducts of the aforementioned reactions. One of skill in the art wouldreadily recognize the compounds that are the products of the reactionsmentioned above. Heretofore the compounds of the invention have beenreferred to by informal or trivial names to convey the scope of themodifications of the invention and that they may occur at one or moresites on the erythropoietin or modified erythropoietin molecule. By waysof non-limiting examples, the following specific compounds are membersof the compound groups embraced herein.

1. Carbamylated erythropoietins: The following compounds representcarbamoyl moieties on the N-terminal amino acid of an erythropoietinmolecule (“alpha-N-carbamoyl-”) or on one (or more) epsilon amino groupsof lysyl residues of erythropoietin (“N-epsilon-carbamoyl-”). Of course,multiple N-epsilon modifications with or without thealpha-N-modification may be present.

-   -   i. alpha-N-carbamoylerythropoietin    -   ii. N-epsilon-carbamoylerythropoietin    -   iii. alpha-N-carbamoyl, N-epsilon-carbamoylerythropoietin    -   iv. alpha-N-carbamoylasialoerythropoietin    -   v. N-epsilon-carbamoylasialoerythropoietin    -   vi. alpha-N-carbamoyl, N-epsilon-carbamoylasialoerythropoietin    -   vii. alpha-N-carbamoylhyposialoerythropoietin    -   viii. N-epsilon-carbamoylhyposialoerythropoietin, and    -   ix. alpha-N-carbamoyl,        N-epsilon-carbamoylhyposialoerythropoietin

2. Succinylated erythropoietins: The following compounds representsuccinyl moieties on the N-terminal amino acid of an erythropoietinmolecule (“alpha-N-succinyl-”) or on one (or more) epsilon amino groupsof lysyl residues of erythropoietin (“N-epsilon-succinyl-”). Of course,multiple N-epsilon modifications with or without thealpha-N-modification may be present.

-   -   i. alpha-N-succinylerythropoietin;    -   ii. N-epsilon-succinylerythropoietin;    -   iii. alpha-N-succinyl, N-epsilon-succinylerythropoietin;    -   iv. alpha-N-succinylasialoerythropoietin;    -   v. N-epsilon-succinylasialoerythropoietin;    -   vi. alpha-N-succinyl, N-epsilon-succinylasialoerythropoietin;    -   vii. alpha-N-succinylhyposialoerythropoietin;    -   viii. N-epsilon-succinylhyposialoerythropoietin; and    -   ix. alpha-N-succinyl, N-epsilon-succinylhyposialoerythropoietin.

3. Acetylated erythropoietins: The following compounds represent acetylmoieties on the N-terminal amino acid of an erythropoietin molecule(“alpha-N-acetyl-”) or on one (or more) epsilon amino groups of lysylresidues of erythropoietin (“N-epsilon-acetyl-”). Of course, multipleN-epsilon modifications with or without the alpha-N-modification may bepresent.

-   -   i. alpha-N-acetylerythropoietin;    -   ii. N-epsilon-acetylerythropoietin;    -   iii. alpha-N-acetyl, N-epsilon-acetylerythropoietin;    -   iv. alpha-N-acetylasialoerythropoietin;    -   v. N-epsilon-acetylasialoerythropoietin;    -   vi. alpha-N-acetyl, N-epsilon-acetylasialoerythropoietin;    -   vii. alpha-N-acetylhyposialoerythropoietin;    -   viii. N-epsilon-acetylhyposialoerythropoietin; and    -   ix. alpha-N-acetyl, N-epsilon-acetylhyposialoerythropoietin.

4. Biotinylated erythropoietins: The following compounds representbiotinyl moieties on the N-terminal amino acid of an erythropoietinmolecule (“alpha-N-biotinyl-”) or on one (or more) epsilon amino groupsof lysyl residues of erythropoietin (“N-epsilon-biotinyl-”). Of course,multiple N-epsilon modifications with or without thealpha-N-modification may be present.

-   -   i. alpha-N-biotinylerythropoietin;    -   ii. N-epsilon-biotinylerythropoietin;    -   iii. alpha-N-biotinyl, N-epsilon-biotinylerythropoietin;    -   iv. alpha-N-biotinylasialoerythropoietin;    -   v. N-epsilon-biotinylasialoerythropoietin;    -   vi. alpha-N-biotinyl, N-epsilon-biotinylasialoerythropoietin;    -   vii. alpha-N-biotinylhyposialoerythropoietin;    -   viii. N-epsilon-biotinylhyposialoerythropoietin; and    -   ix. alpha-N-biotinyl, N-epsilon-biotinylhyposialoerythropoietin.

5. Iodinated erythropoietins: Of course, one of ordinary skill in theart would recognize that several different tyrosine residues as well ascombinations of tyrosine residues within erythropoietin may be iodinatedand that ones provided are merely illustrative.

-   -   i. Iodoerythropoietin;    -   ii. Iodoasialoerythropoietin; and    -   iii. Iodohyposialoerythropoietin.

6. Carboxymethyllysyl-erythropoietins: The following compounds representcarboxymethyl moieties on one (or more) epsilon amino groups of lysylresidues of erythropoietin (“N-epsilon-carboxymethyl-”). Of course,multiple N-epsilon modifications may be present.

-   -   i. N-epsilon-carboxymethylerythropoietin;    -   ii. N-epsilon-carboxymethylasialoerythropoietin; and    -   iii. N-epsilon-carboxymethylhyposialoerythropoietin.

A variety of host-expression vector systems may be utilized to producethe erythropoietins and erythropoietin-related molecules of theinvention. Such host-expression systems represent vehicles by which theerythropoietins of interest may be produced and subsequently purified,but also represent cells that may, when transformed or transfected withthe appropriate nucleotide coding sequences, exhibit the modifiederythropoietin gene product in situ. These include but are not limitedto, bacteria, insect, plant, mammalian, including human host systems,such as, but not limited to, insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing themodified erythropoietin product coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containingerythropoietin-related molecule coding sequences; or mammalian cellsystems, including human cell systems, (e.g., HT1080, COS, CHO, BHK,293, 3T3) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells, including human host cells, include but are not limited toHT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theerythropoietin-related molecule gene product may be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci that in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell linesthat express the erythropoietin-related molecule gene product. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the endogenous activity of theerythropoietin-related molecule gene product.

Alternatively, the expression characteristic of an endogenouserythropoietin gene within a cell line or microorganism may be modifiedby inserting a heterologous DNA regulatory element into the genome of astable cell line or cloned microorganism such that the insertedregulatory element is operatively linked with the endogenouserythropoietin gene. For example, an endogenous erythropoietin genewhich is normally “transcriptionally silent,” i.e., an erythropoietingene which is normally not expressed, or is expressed only a very lowlevels in a cell line, may be activated by inserting a regulatoryelement which is capable of promoting the expression of a normallyexpressed gene product in that cell line or microorganism.Alternatively, a transcriptionally silent, endogenous erythropoietingene may be activated by insertion of a promiscuous regulatory elementthat works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous erythropoietin gene, using techniques, such as targetedhomologous recombination, which are well known to those of skill in theart, and described e.g., in French Patent No. 2646438 to InstitutPasteur, U.S. Pat. No. 4,215,051 to Chappel; U.S. Pat. No. 5,578,461 toSherwin et al.; International Application No. PCT/US92/09627(WO93/09222) by Selden et al.; and International Application No.PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which isincorporated by reference herein in its entirety.

In one embodiment of the invention, a tissue protective cytokine is achemically modified erythropoietin molecule deficient in sialicresidues, or completely lacking sialic residues, and may be produced inmammalian cell, including a human cell. Such cells may be engineered tobe deficient in, or lacking, the enzymes that add sialic acids, i.e.,the β-galactoside α 2,3 sialyltransferase (Aα 2,3 sialyltransferase@)and the β-galactoside a 2,6 sialyltransferase (Aα 2,6sialyltransferase@) activity. In one embodiment, a mammalian cell isused in which either or both the α 2,3 sialyltransferase gene and/or theα 2,6 sialyltransferase gene, is deleted. Such deletions may beconstructed using gene knock-out techniques well known in the art. Inanother embodiment, dihydrofolate reductase (DHFR) deficient ChineseHamster Ovary (CHO) cells are used as the host cell for the productionof recombinant erythropoietin-related molecules. CHO cells do notexpress the enzyme α 2,6 sialyltransferase and therefore do not addsialic acid in the 2,6 linkage to N-linked oligosaccharides ofglycoproteins produced in these cells. As a result, recombinant proteinsproduced in CHO cells lack sialic acid in the 2,6 linkage to galactose(Sasaki et al. (1987; Takeuchi et al. supra; Mutsaers et al Eur. J.Biochem. 156, 651 (1986); Takeuchi et al. J. Chromotgr. 400, 207 (1987).In one embodiment, to produce a host cell for the production ofasialo-erythropoietin, the gene encoding α 2, 3 sialyltransferase in CHOcells is deleted. Such α 2, 3 sialyltransferase knock-out CHO cellscompletely lack sialyltransferase activity, and as a result, are usefulfor the recombinant expression and production of a tissue protectivecytokine consists of asialo-erythropoietin.

In another embodiment, asialo glycoproteins can be produced byinterfering with sialic acid transport into the Golgi apparatus e.g.,Eckhardt et al., 1998, J. Biol. Chem. 273:20189-95). Using methods wellknown to those skilled in the art (e.g., Oelmann et al., 2001, J. Biol.Chem. 276:26291-300), mutagenesis of the nucleotide sugar CMP-sialicacid transporter can be accomplished to produce mutants of Chinesehamster ovary cells. These cells cannot add sialic acid residues toglycoproteins such as erythropoietin and produce onlyasialoerythropoietin.

Transfected mammalian cells producing erythropoietin also producecytosolic sialidase which if it leaks into the culture medium degradessialoerythropoietin with high efficiency (e.g., Gramer et al, 1995Biotechnology 13:692-698). Using methods well known to thoseknowledgeable in the art (e.g., from information provided in Ferrari etal, 1994, Glycobiology 4:367-373), cell lines can be transfected,mutated or otherwise caused to constitutively produce sialidase. In thismanner, asialoerythropoietin can be produced during the manufacture ofasialoerythropoietin.

A tissue protective cytokine of the invention has at least onemodification of an amino acid residue in erythropoietin, regardless ofthe glycosylation state of the molecule. As mentioned above, thechemical modification may be at least a modification of at least oneamino group of at least one amino acid, such as a lysine residue, or theN-terminal amino group, or iodination of at least one tyrosine residue.

Following the manufacture of the recombinant tissue protective cytokinesand chemically modified recombinant tissue protective cytokines of thepresent invention, one of ordinary skill in the art can verify thetissue protective attributes of the cytokines and the absence of aneffect on the bone marrow using well known assays.

For example, the non-erythropoietic affect of a recombinant tissueprotective cytokine can be verified through the use of a TF-1 assay. Inthis assay TF-1 cells are grown in a complete RPMI medium supplementedwith 5 ng/ml of GM-CSF and 10% FCS for a day at 37 C in a CO₂ incubator.The cells are then washed in and suspended at a density of 10⁶ cells/mlfor 16 hours in starvation medium (5% FCS without GM-CSF). A 96 wellplate is prepared by: (1) adding 100 μl of sterile water to the outerwells to maintain moisture; (2) adding medium (10% FCS without cells orGM-CSF) alone to 5 wells; and (3) seeding 25,000 cells/well with mediumcontaining 10% FCS and the recombinant tissue protective cytokines inremaining wells (five wells per cytokine being tested). If the cellsproliferate, the recombinant tissue protective cytokine may beerythropoietic. The in vivo affect of the compound should then be testedon an in vivo assay monitoring an increase of hematocrit due to therecombinant tissue protective cytokine A negativeresult—non-proliferation of cells in the TF-1 assay in vitro assay or noincrease in hematocrit within the in vivo assay—means that therecombinant tissue protective cytokine is nonerythropoietic.

The tissue protective properties of the recombinant tissue protectivecytokine may be verified using a P-19 in vitro assay or a waterintoxication in vivo assay in rats, both of which are outlined infurther detail below. The above assays are provided merely as examples,and other suitable assays to determine the effect of the cytokines onbone marrow and tissue protection are known to those of ordinary skillin the art are contemplated by the present invention as well.

In the practice of one aspect of the present invention, a pharmaceuticalcomposition as described above containing a tissue protective cytokinemay be administrable to a mammal by any route which provides asufficient level of a tissue protective cytokine in the vasculature topermit translocation across an endothelial cell barrier and beneficialeffects on responsive cells. When used for the purpose of perfusing atissue or organ, similar results are desired. In the instance whereinthe tissue protective cytokine is used for ex-vivo perfusion, the tissueprotective cytokine may be any of the aforementioned chemically-modifiederythropoietins. In the instance where the cells or tissue isnon-vascularized and/or the administration is by bathing the cells ortissue with the composition of the invention, the pharmaceuticalcomposition provides an effective responsive-cell-beneficial amount of atissue protective cytokine. The endothelial cell barriers across which atissue protective cytokine may translocate include tight junctions,perforated junctions, fenestrated junctions, and any other types ofendothelial barriers present in a mammal. A preferred barrier is anendothelial cell tight junction, but the invention is not so limiting.

The aforementioned tissue protective cytokines are useful generally forthe therapeutic or prophylactic treatment of human diseases of thecentral nervous system or peripheral nervous system which have primarilyneurological or psychiatric symptoms, ophthalmic diseases,cardiovascular diseases, cardiopulmonary diseases, respiratory diseases,kidney, urinary and reproductive diseases, bone diseases, skin diseases,gastrointestinal diseases and endocrine and metabolic abnormalities. Inparticular, such conditions and diseases include hypoxic conditions,which adversely affect excitable tissues, such as excitable tissues inthe central nervous system tissue, peripheral nervous system tissue, orcardiac tissue or retinal tissue such as, for example, brain, heart, orretina/eye. Therefore, the invention can be used to treat or preventdamage to excitable tissue resulting from hypoxic conditions in avariety of conditions and circumstances. Non-limiting examples of suchconditions and circumstances are provided in the table herein below.

In the example of the protection of neuronal tissue pathologiestreatable in accordance with the present invention, such pathologiesinclude those which result from reduced oxygenation of neuronal tissues.Any condition which reduces the availability of oxygen to neuronaltissue, resulting in stress, damage, and finally, neuronal cell death,can be treated by the methods of the present invention. Generallyreferred to as hypoxia and/or ischemia, these conditions arise from orinclude, but are not limited to stroke, vascular occlusion, prenatal orpostnatal oxygen deprivation, suffocation, choking, near drowning,carbon monoxide poisoning, smoke inhalation, trauma, including surgeryand radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructivepulmonary disease, emphysema, adult respiratory distress syndrome,hypotensive shock, septic shock, anaphylactic shock, insulin shock,sickle cell crisis, cardiac arrest, dysrhythmia, nitrogen narcosis, andneurological deficits caused by heart-lung bypass procedures.

In one embodiment, for example, the specific tissue protective cytokinecompositions can be administered to prevent injury or tissue damageresulting from risk of injury or tissue damage during surgicalprocedures, such as, for example, tumor resection or aneurysm repair.Other pathologies caused by or resulting from hypoglycemia which aretreatable by the methods described herein include insulin overdose, alsoreferred to as iatrogenic hyperinsulinemia, insulinoma, growth hormonedeficiency, hypocortisolism, drug overdose, and certain tumors.

Other pathologies resulting from excitable neuronal tissue damageinclude seizure disorders, such as epilepsy, convulsions, or chronicseizure disorders. Other treatable conditions and diseases includediseases such as stroke, multiple sclerosis, hypotension, cardiacarrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brainor spinal cord trauma, AIDS dementia, age-related loss of cognitivefunction, memory loss, amyotrophic lateral sclerosis, seizure disorders,alcoholism, retinal ischemia, optic nerve damage resulting fromglaucoma, and neuronal loss.

The specific composition and methods of the present invention may beused to treat inflammation resulting from disease conditions or varioustraumas, such as physically or chemically induced inflammation. Suchtraumas could include angitis, chronic bronchitis, pancreatitis,osteomyelitis, rheumatoid arthritis, glomerulonephritis, optic neuritis,temporal arteritis, encephalitis, meningitis, transverse myelitis,dermatomyositis, polymyositis, necrotizing fascilitis, hepatitis, andnecrotizing enterocolitis.

Evidence has demonstrated that activated astrocytes can exert acytotoxic role towards neurons by producing neurotoxins. Nitric oxide,reactive oxygen species, and cytokines are released from glial cells inresponse to cerebral ischemia (see Becker, K. J. 2001. Targeting thecentral nervous system inflammatory response in ischemic stroke. CurrOpinion Neurol 14:349-353 and Mattson, M. P., Culmsee, C., and Yu, Z. F.2000. Apoptotic and Antiapoptotic mechanisms in stroke. Cell TissueRes301:173-187.). Studies have further demonstrated that in models ofneurodegeneration, glial activation and subsequent production ofinflammatory cytokines depends upon primary neuronal damage (seeViviani, B., Corsini, E., Galli, C. L., Padovani, A., Ciusani, E., andMarinovich, M. 2000. Dying neural cells activate glia through therelease of a protease product. Glia 32:84-90 and Rabuffetti, M.,Scioratti, C., Tarozzo, G., Clementi, E., Manfredi, A. A., and Beltramo,M. 2000. Inhibition of caspase-1-like activity byAc-Tyr-Val-Ala-Asp-chloromethyl ketone includes long lastingneuroprotection in cerebral ischemia through apoptosis reduction anddecrease of proinflammatory cytokines J Neurosci 20:4398-4404).Inflammation and glial activation is common to different forms of neurodegenerative disorders, including cerebral ischemia, brain trauma andexperimental allergic encephalomyelitis, disorders in whicherythropoietin exerts a neuroprotective effect. Inhibition of cytokineproduction by erythropoietin could, at least in part, mediate itsprotective effect. However, unlike “classical” anti-inflammatorycytokines such as Il-10 and IL-13, which inhibit tumor necrosis factorproduction directly, erythropoietin appears to be active only in thepresence of neuronal death.

While not wishing to be bound by any particular theory it appears thatthis anti-inflammatory activity may be hypothetically explained byseveral non-limiting theories. First, since erythropoietin preventsapoptosis, inflammatory events triggered by apoptosis would beprevented. Additionally, erythropoietin may prevent the release ofmolecular signals from dying neurons which stimulate the glia cells orcould act directly on the glial cells reducing their reaction to theseproducts. Another possibility is that erythropoietin targets moreproximal members of the inflammatory cascade (e.g., caspase 1, reactiveoxygen or nitrogen intermediates) that trigger both apoptosis andinflammation.

Furthermore, erythropoietin appears to provide anti-inflammatoryprotection without the rebound affect typically associated with otheranti-inflammatory compounds such as dexamethasone. Once again, notwishing to be bound by any particular theory, it appears as though thismay be due to erythropoietin's affect on multipurpose neuro toxins suchas nitric oxide (NO). Although activated astrocytes and microgliaproduce neurotoxic quantities of NO in response to various traumas, NOserves many purposes within the body including the modulation ofessential physiological functions. Thus, although the use of ananti-inflammatory may alleviate inflammation by suppressing NO or otherneuro toxins, if the anti-inflammatory has too long a half-life it mayalso interfere with these chemical's roles in repairing the damageresulting from the trauma that led to the inflammation. It ishypothesized that the tissue protective cytokines of the presentinvention are able to alleviate the inflammation without interferingwith the restorative capabilities of neurotoxins such as NO.

The specific compositions and methods of the invention may be used totreat conditions of, and damage to, retinal tissue. Such disordersinclude, but are not limited to retinal ischemia, macular degeneration,retinal detachment, retinitis pigmentosa, arteriosclerotic retinopathy,hypertensive retinopathy, retinal artery blockage, retinal veinblockage, hypotension, and diabetic retinopathy.

In another embodiment, the methods and principles of the invention maybe used to protect or treat injury resulting from radiation damage toexcitable tissue. A further utility of the methods of the presentinvention is in the treatment of neurotoxin poisoning, such as domoicacid shellfish poisoning, neurolathyrism, and Guam disease, amyotrophiclateral sclerosis, and Parkinson's disease.

As mentioned above, the present invention is also directed to a methodfor enhancing excitable tissue function in a mammal by peripheraladministration of a tissue protective cytokine as described above.Various diseases and conditions are amenable to treatment using thismethod, and further, this method is useful for enhancing cognitivefunction in the absence of any condition or disease. These uses of thepresent invention are describe in further detail below and includeenhancement of learning and training in both human and non-humanmammals.

Conditions and diseases treatable by the methods of this aspect of thepresent invention directed to the central nervous system include but arenot limited to mood disorders, anxiety disorders, depression, autism,attention deficit hyperactivity disorder, and cognitive dysfunction.These conditions benefit from enhancement of neuronal function. Otherdisorders treatable in accordance with the teachings of the presentinvention include sleep disruption, for example, sleep apnea andtravel-related disorders; subarachnoid and aneurismal bleeds,hypotensive shock, concussive injury, septic shock, anaphylactic shock,and sequelae of various encephalitides and meningitides, for example,connective tissue disease-related cerebritides such as lupus. Other usesinclude prevention of or protection from poisoning by neurotoxins, suchas domoic acid shellfish poisoning, neurolathyrism, and Guam disease,amyotrophic lateral sclerosis, Parkinson's disease; postoperativetreatment for embolic or ischemic injury; whole brain irradiation;sickle cell crisis; and eclampsia.

A further group of conditions treatable by the methods of the presentinvention include mitochondrial dysfunction, of either a hereditary oracquired nature, which are the cause of a variety of neurologicaldiseases typified by neuronal injury and death. For example, Leighdisease (subacute necrotizing encephalopathy) is characterized byprogressive visual loss and encephalopathy, due to neuronal drop out,and myopathy. In these cases, defective mitochondrial metabolism failsto supply enough high energy substrates to fuel the metabolism ofexcitable cells. An erythropoietin receptor activity modulator optimizesfailing function in a variety of mitochondrial diseases. As mentionedabove, hypoxic conditions adversely affect excitable tissues. Theexcitable tissues include, but are not limited to, central nervoussystem tissue, peripheral nervous system tissue, and heart tissue. Inaddition to the conditions described above, the methods of the presentinvention are useful in the treatment of inhalation poisoning such ascarbon monoxide and smoke inhalation, severe asthma, adult respiratorydistress syndrome, and choking and near drowning. Further conditionswhich create hypoxic conditions or by other means induce excitabletissue damage include hypoglycemia that may occur in inappropriatedosing of insulin, or with insulin-producing neoplasms (insulinoma).

Various neuropsychologic disorders which are believed to originate fromexcitable tissue damage are treatable by the instant methods. Chronicdisorders in which neuronal damage is involved and for which treatmentby the present invention is provided include disorders relating to thecentral nervous system and/or peripheral nervous system includingage-related loss of cognitive function and senile dementia, chronicseizure disorders, Alzheimer's disease, Parkinson's disease, dementia,memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberoussclerosis, Wilson's Disease, cerebral and progressive supranuclearpalsy, Guam disease, Lewy body dementia, prion diseases, such asspongiform encephalopathies, e.g., Creutzfeldt-Jakob disease,Huntington's disease, myotonic dystrophy, Freidrich's ataxia and otherataxias, as well as Gilles de la Tourette's syndrome, seizure disorderssuch as epilepsy and chronic seizure disorder, stroke, brain or spinalcord trauma, AIDS dementia, alcoholism, autism, retinal ischemia,glaucoma, autonomic function disorders such as hypertension and sleepdisorders, and neuropsychiatric disorders that include, but are notlimited to schizophrenia, schizoaffective disorder, attention deficitdisorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, panic disorder, as well as unipolar and bipolar affectivedisorders. Additional neuropsychiatric and neurodegenerative disordersinclude, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which in incorporated herein byreference in its entirety.

In another embodiment, recombinant chimeric toxin molecules comprisingerythropoietin can be used for therapeutic delivery of toxins to treat aproliferative disorder, such as cancer, or viral disorder, such assubacute sclerosing panencephalitis.

The following table lists additional exemplary, non-limiting indicationsas to the various conditions and diseases amenable to treatment by theaforementioned tissue protective cytokines

Cell, tissue or Dysfunction or organ pathology Condition or disease TypeHeart Ischemia Coronary artery Acute, chronic disease Stable, unstableMyocardial Dressler's syndrome infarction Angina Congenital heartValvular disease Cardiomyopathy Prinzmetal angina Cardiac ruptureAneurysmatic Septal perforation Angiitis Arrhythmia Tachy-, Stable,unstable bradyarrhythmia Hypersensitive carotid sinus Supraventricular,node ventricular Conduction abnormalities Congestive heart Left, right,bi- Cardiomyopathies, such as failure ventricular idiopathic familial,infective, metabolic, storage disease, deficiencies, connective tissuedisorder, infiltration and granulomas, neurovascular MyocarditisAutoimmune, infective, idiopathic Cor pulmonale Blunt and penetratingtrauma Toxins Cocaine Vascular Hypertension Primary, secondaryDecompression sickness Fibromuscular hyperplasia Aneurysm Dissecting,ruptured, enlarging Lungs Obstructive Asthma Chronic bronchitis,Emphysema and airway obstruction Ischemic lung disease Pulmonaryembolism, Pulmonary thrombosis, Fat embolism Environmental lung diseasesIschemic lung disease Pulmonary embolism Pulmonary thrombosisInterstitial lung Idiopathic pulmonary disease fibrosis CongenitalCystic fibrosis Cor pulmonale Trauma Pneumonia and Infectious,parasitic, pneumonitides toxic, traumatic, burn, aspiration SarcoidosisPancreas Endocrine Diabetes mellitus, Beta cell failure, dysfunctiontype I and II Diabetic neuropathy Other endocrine cell failure of thepancreas Exocrine Exocrine pancreas Pancreatitis failure Bone OsteopeniaPrimary Hypogonadism immobilisation secondary Postmenopausal Age-relatedHyperparathyroidism Hyperthyroidism Calcium, magnesium, phosphorusand/or vitamin D deficiency Osteomyelitis Avascular necrosis TraumaPaget's disease Skin Alopecia Areata Primary Totalis Secondary Malepattern baldness Vitiligo Localized Primary Generalized SecondaryDiabetic ulceration Peripheral vascular disease Burn injuries AutoimmuneLupus erythematodes, disorders Sjiogren, Rheumatoid arthritis,Glomerulonephritis, Angiitis Langerhan's histiocytosis Eye Opticneuritis Blunt and penetrating injuries, Infections, Sarcoid, Sickle Cdisease, Retinal detachment, Temporal arteritis Retinal ischemia,macular degeneration, retinal detachment, retinitis pigmentosa,arteriosclerotic retinopathy, hypertensive retinopathy, retinal arteryblockage, retinal vein blockage, hypotension, and diabetic retinopathy.Embryonic and Asphyxia fetal disorders Ischemia CNS Chronic fatiguesyndrome, acute and chronic hypoosmolar and hyperosmolar syndromes, AIDSDementia, Electrocution Encephalitis Rabies, Herpes Meningitis Subduralhematoma Nicotine addiction Drug abuse and Cocaine, heroin, withdrawalcrack, marijuana, LSD, PCP, poly-drug abuse, ecstasy, opioids, sedativehypnotics, amphetamines, caffeine Obsessive- compulsive disorders Spinalstenosis, Transverse myelitis, Guillian Barre, Trauma, Nerve rootcompression, Tumoral compression, Heat stroke ENT Tinnitus Meuniere'ssyndrome Hearing loss Traumatic injury, barotrauma Kidney Renal failureAcute, chronic Vascular/ischemic, interstitial disease, diabetic kidneydisease, nephrotic syndromes, infections Henoch S. Purpura Striatedmuscle Autoimmune Myasthenia gravis disorders DermatomyositisPolymyositis Myopathies Inherited metabolic, endocrine and toxic Heatstroke Crush injury Rhabdomylosis Mitochondrial disease InfectionNecrotizing fasciitis Sexual Central and Impotence secondary dysfunctionperipheral to medication Liver Hepatitis Viral, bacterial, parasiticIschemic disease Cirrhosis, fatty liver Infiltrative/metabolic diseasesGastrointestinal Ischemic bowel disease Inflammatory bowel diseaseNecrotizing enterocolitis Organ Treatment of donor transplantation andrecipient Reproductive Infertility Vascular tract Autoimmune Uterineabnormalities Implantation disorders Endocrine Glandular hyper- andhypofunction

As mentioned above, these diseases, disorders or conditions are merelyillustrative of the range of benefits provided by the tissue protectivecytokines of the invention. Accordingly, this invention generallyprovides therapeutic or prophylactic treatment of the consequences ofmechanical trauma or of human diseases. Therapeutic or prophylactictreatment for diseases, disorders or conditions of the CNS and/orperipheral nervous system are preferred. Therapeutic or prophylactictreatment for diseases, disorders or conditions which have a psychiatriccomponent is provided. Therapeutic or prophylactic treatment fordiseases, disorders or conditions including but not limited to thosehaving an ophthalmic, cardiovascular, cardiopulmonary, respiratory,kidney, urinary, reproductive, gastrointestinal, endocrine, or metaboliccomponent is provided.

One of ordinary skill in the art would understand that thepharmaceutical composition of the present invention may be made of amixture of the tissue protective cytokines of the present invention aswell as erythropoietin.

In one embodiment, such a pharmaceutical composition of erythropoietinor tissue protective cytokine may be administered systemically toprotect or enhance the target cells, tissue or organ. Suchadministration may be parenterally, via inhalation, or transmucosally,e.g., orally, nasally, rectally, intravaginally, sublingually,submucosally or transdermally. Preferably, administration is parenteral,e.g., via intravenous or intraperitoneal injection, and also including,but is not limited to, intra-arterial, intramuscular, intradermal andsubcutaneous administration.

For other routes of administration, such as by use of a perfusate,injection into an organ, or other local administration, a pharmaceuticalcomposition will be provided which results in similar levels of a tissueprotective cytokine as described above. A level of about 15 pM-30 nM ispreferred.

The pharmaceutical compositions of the invention may comprise atherapeutically effective amount of a compound, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized foreign pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions in water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. The compounds ofthe invention can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for oral administration may beprovided as capsules or tablets; as powders or granules; as solutions,syrups or suspensions (in aqueous or non-aqueous liquids); as ediblefoams or whips; or as emulsions. Tablets or hard gelatine capsules maycomprise lactose, starch or derivatives thereof, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, stearic acid or saltsthereof. Soft gelatine capsules may comprise vegetable oils, waxes,fats, semi-solid, or liquid polyols etc. Solutions and syrups maycomprise water, polyols and sugars.

An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract (e.g., glycerylmonostearate or glyceryl distearate may be used). Thus, the sustainedrelease of an active agent may be achieved over many hours and, ifnecessary, the active agent can be protected from being degraded withinthe stomach. Pharmaceutical compositions for oral administration may beformulated to facilitate release of an active agent at a particulargastrointestinal location due to specific pH or enzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.Pharmaceutical compositions adapted for topical administration may beprovided as ointments, creams, suspensions, lotions, powders, solutions,pastes, gels, sprays, aerosols or oils. For topical administration tothe skin, mouth, eye or other external tissues a topical ointment orcream is preferably used. When formulated in an ointment, the activeingredient may be employed with either a paraffinic or a water-miscibleointment base. Alternatively, the active ingredient may be formulated ina cream with an oil-in-water base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops. In these compositions, the active ingredient can be dissolved orsuspended in a suitable carrier, e.g., in an aqueous solvent.Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouthwashes.

Pharmaceutical compositions adapted for nasal and pulmonaryadministration may comprise solid carriers such as powders (preferablyhaving a particle size in the range of 20 to 500 microns). Powders canbe administered in the manner in which snuff is taken, i.e., by rapidinhalation through the nose from a container of powder held close to thenose. Alternatively, compositions adopted for nasal administration maycomprise liquid carriers, e.g., nasal sprays or nasal drops.Alternatively, inhalation of compounds directly into the lungs may beaccomplished by inhalation deeply or installation through a mouthpieceinto the oropharynx. These compositions may comprise aqueous or oilsolutions of the active ingredient. Compositions for administration byinhalation may be supplied in specially adapted devices including, butnot limited to, pressurized aerosols, nebulizers or insufflators, whichcan be constructed so as to provide predetermined dosages of the activeingredient. In a preferred embodiment, pharmaceutical compositions ofthe invention are administered into the nasal cavity directly or intothe lungs via the nasal cavity or oropharynx.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which may contain antioxidants, buffers, bacteriostats andsolutes that render the compositions substantially isotonic with theblood of an intended recipient. Other components that may be present insuch compositions include water, alcohols, polyols, glycerine andvegetable oils, for example. Compositions adapted for parenteraladministration may be presented in unit-dose or multi-dose containers,for example sealed ampules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of asterile liquid carrier, e.g., sterile saline solution for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.In one embodiment, an autoinjector comprising an injectable solution ofan erythropoietin may be provided for emergency use by ambulances,emergency rooms, and battlefield situations, and even forself-administration in a domestic setting, particularly where thepossibility of traumatic amputation may occur, such as by imprudent useof a lawn mower. The likelihood that cells and tissues in a severed footor toe will survive after reattachment may be increased by administeringerythropoietin or a tissue protective cytokine to multiple sites in thesevered part as soon as practicable, even before the arrival of medicalpersonnel on site, or arrival of the afflicted individual with severedtoe in tow at the emergency room.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water-free concentrate in a hermetically-sealedcontainer such as an ampule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampule of sterile saline can be providedso that the ingredients may be mixed prior to administration.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

A perfusate composition may be provided for use in transplanted organbaths, for in situ perfusion, or for administration to the vasculatureof an organ donor prior to organ harvesting. Such pharmaceuticalcompositions may comprise levels of erythropoietin, tissue protectivecytokines, or a form of either erythropoietin or tissue protectivecytokines not suitable for acute or chronic, local or systemicadministration to an individual, but will serve the functions intendedherein in a cadaver, organ bath, organ perfusate, or in situ perfusateprior to removing or reducing the levels of the erythropoietin containedtherein before exposing or returning the treated organ or tissue toregular circulation. The erythropoietin for this aspect of the inventionmay be any erythropoietin, such as naturally-occurring forms such ashuman erythropoietin, or any of tissue protective cytokines hereinabovedescribed, such as asialoerythropoietin andphenylglyoxal-erythropoietins, as non-limiting examples.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In another embodiment, for example, a tissue protective cytokine can bedelivered in a controlled-release system. For example, the polypeptidemay be administered using intravenous infusion, an implantable osmoticpump, a transdermal patch, liposomes, or other modes of administration.In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987,CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In anotherembodiment, the compound can be delivered in a vesicle, in particular aliposome (see Langer, Science 249:1527-1533 (1990); Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);WO 91/04014; U.S. Pat. No. 4,704,355; Lopez-Berestein, ibid., pp.317-327; see generally ibid.). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Press: Boca Raton, Fla., 1974; ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol.Sci. Rev. Macromol. Chem. 23:61, 1953; see also Levy et al., 1985,Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard etal., 1989, J. Neurosurg. 71:105).

In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the target cells, tissue ororgan, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, pp. 115-138 in Medical Applications of Controlled Release, vol.2, supra, 1984). Other controlled release systems are discussed in thereview by Langer (1990, Science 249:1527-1533).

In another embodiment, a tissue protective cytokine, as properlyformulated, can be administered by nasal, oral, rectal, vaginal, orsublingual administration.

In a specific embodiment, it may be desirable to administererythropoietin and/or the tissue protective cytokines of the inventionlocally to the area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion during surgery,topical application, e.g., in conjunction with a wound dressing aftersurgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material, including membranes, such assilastic membranes, or fibers.

Selection of the preferred effective dose will be readily determinableby a skilled artisan based upon considering several factors, which willbe known to one of ordinary skill in the art. Such factors include theparticular form of erythropoietin or the tissue protective cytokine, andits pharmacokinetic parameters such as bioavailability, metabolism,half-life, etc., which will have been established during the usualdevelopment procedures typically employed in obtaining regulatoryapproval for a pharmaceutical compound. Further factors in consideringthe dose include the condition or disease to be treated or the benefitto be achieved in a normal individual, the body mass of the patient, theroute of administration, whether administration is acute or chronic,concomitant medications, and other factors well known to affect theefficacy of administered pharmaceutical agents. Thus the precise dosageshould be decided according to the judgment of the practitioner and eachpatient's circumstances, e.g., depending upon the condition and theimmune status of the individual patient, and according to standardclinical techniques.

In another aspect of the invention, a perfusate or perfusion solution isprovided for perfusion and storage of organs for transplant, theperfusion solution includes an amount of erythropoietin or a tissueprotective cytokine effective to protect responsive cells and associatedcells, tissues or organs. Transplant includes but is not limited toxenotransplantation, where an organ (including cells, tissue or otherbodily part) is harvested from one donor and transplanted into adifferent recipient; and autotransplant, where the organ is taken fromone part of a body and replaced at another, including bench surgicalprocedures, in which an organ may be removed, and while ex vivo,resected, repaired, or otherwise manipulated, such as for tumor removal,and then returned to the original location. In one embodiment, theperfusion solution is the University of Wisconsin (UW) solution (U.S.Pat. No. 4,798,824) which contains from about 1 to about 25 U/mlerythropoietin, 5% hydroxyethyl starch (having a molecular weight offrom about 200,000 to about 300,000 and substantially free of ethyleneglycol, ethylene chlorohydrin, sodium chloride and acetone); 25 mMKH₂PO₄; 3 mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM HEPESbuffer; 5 mM magnesium gluconate; 1.5 mM CaCl₂; 105 mM sodium gluconate;200,000 units penicillin; 40 units insulin; 16 mg dexamethasone; 12 mgPhenol Red; and has a pH of 7.4-7.5 and an osmolality of about 320mOsm/l. The solution is used to maintain cadaveric kidneys andpancreases prior to transplant. Using the solution, preservation can beextended beyond the 30-hour limit recommended for cadaveric kidneypreservation. This particular perfusate is merely illustrative of anumber of such solutions that can be adapted for the present use byinclusion of an effective amount of erythropoietin and/or a tissueprotective cytokine. In a further embodiment, the perfusate solutioncontains from about 1 to about 500 ng/ml erythropoietin, or from about40 to about 320 ng/ml erythropoietin. As mentioned above, any form oferythropoietin or tissue protective cytokines can be used in this aspectof the invention.

While the preferred recipient of a tissue protective cytokine for thepurposes herein throughout is a human, the methods herein apply equallyto other mammals, particularly domesticated animals, livestock,companion, and zoo animals. However, the invention is not so limitingand the benefits can be applied to any mammal.

In further aspects of the ex-vivo invention, erythropoietin and anytissue protective cytokine such as but not limited to the ones describedabove may be employed.

In another aspect of the invention, methods and compositions forenhancing the viability of cells, tissues or organs which are notisolated from the vasculature by an endothelial cell barrier areprovided by exposing the cells, tissue or organs directly to apharmaceutical composition comprising erythropoietin or a tissueprotective cytokine, or administering or contacting a pharmaceuticalcomposition containing erythropoietin or a tissue protective cytokine tothe vasculature of the tissue or organ. Enhanced activity of responsivecells in the treated tissue or organ is responsible for the positiveeffects exerted.

As described above, the invention is based, in part, on the discoverythat erythropoietin molecules can be transported from the luminalsurface to the basement membrane surface of endothelial cells of thecapillaries of organs with endothelial cell tight junctions, including,for example, the brain, retina, and testis. Thus, responsive cellsacross the barrier are susceptible targets for the beneficial effects oferythropoietin or tissue protective cytokines, and others cell types ortissues or organs that contain and depend in whole or in part onresponsive cells therein are targets for the methods of the invention.While not wishing to be bound by any particular theory, aftertranscytosis of erythropoietin or the tissue protective cytokine,erythropoietin or the tissue protective cytokine can interact with anerythropoietin receptor on a responsive cell, for example, neuronal,retinal, muscle, heart, lung, liver, kidney, small intestine, adrenalcortex, adrenal medulla, capillary endothelial, testes, ovary, orendometrial cell, and receptor binding can initiate a signaltransduction cascade resulting in the activation of a gene expressionprogram within the responsive cell or tissue, resulting in theprotection of the cell or tissue, or organ, from damage, such as bytoxins, chemotherapeutic agents, radiation therapy, hypoxia, etc. Thus,methods for protecting responsive cell-containing tissue from injury orhypoxic stress, and enhancing the function of such tissue are describedin detail herein below.

In the practice of one embodiment of the invention, a mammalian patientis undergoing systemic chemotherapy for cancer treatment, includingradiation therapy, which commonly has adverse effects such as nerve,lung, heart, ovarian or testicular damage. Administration of apharmaceutical composition comprising erythropoietin and/or a tissueprotective cytokine as described above is performed prior to and duringchemotherapy and/or radiation therapy, to protect various tissues andorgans from damage by the chemotherapeutic agent, such as to protect thetestes. Treatment may be continued until circulating levels of thechemotherapeutic agent have fallen below a level of potential danger tothe mammalian body.

In the practice of another embodiment of the invention, various organswere planned to be harvested from a victim of an automobile accident fortransplant into a number of recipients, some of which required transportfor an extended distance and period of time. Prior to organ harvesting,the victim was infused with a pharmaceutical composition comprisingerythropoietin and/or tissue protective cytokines as described herein.Harvested organs for shipment were perfused with a perfusate containingerythropoietin and/or tissue protective cytokines as described herein,and stored in a bath comprising erythropoietin and/or tissue protectivecytokines Certain organs were continuously perfused with a pulsatileperfusion device, utilizing a perfusate containing erythropoietin and/ortissue protective cytokines in accordance with the present invention.Minimal deterioration of organ function occurred during the transportand upon implant and reperfusion of the organs in situ.

In another embodiment of the invention, a surgical procedure to repair aheart valve required temporary cardioplegia and arterial occlusion.Prior to surgery, the patient was infused with a tissue protectivecytokine, 4 μg of carbamylated asialoerythropoietin per kg body weight.Such treatment prevented hypoxic ischemic cellular damage, particularlyafter reperfusion.

In another embodiment of the invention, in any surgical procedure, suchas in cardiopulmonary bypass surgery, a naturally-occurringerythropoietin or a tissue protective cytokine of the invention can beused. In one embodiment, administration of a pharmaceutical compositioncomprising erythropoietin and/or tissue protective cytokines asdescribed above is performed prior to, during, and/or following thebypass procedure, to protect the function of brain, heart, and otherorgans.

In the foregoing examples in which naturally-occurring erythropoietinand/or a tissue protective cytokine of the invention is used for ex-vivoapplications, or to treat responsive cells such as neuronal tissue,retinal tissue, heart, lung, liver, kidney, small intestine, adrenalcortex, adrenal medulla, capillary endothelial, testes, ovary, orendometrial cells or tissue, the invention provides a pharmaceuticalcomposition in dosage unit form adapted for protection or enhancement ofresponsive cells, tissues or organs distal to the vasculature whichcomprises an amount within the range from about 1 pg to 5 mg, 500 pg to5 mg, 1 ng to 5 mg, 500 ng to 5 mg, 1 μg to 5 mg, 500 μg to 5 mg, or 1mg to 5 mg of a tissue protective cytokine, and a pharmaceuticallyacceptable carrier. In a preferred embodiment, the amount of tissueprotective cytokine is within the range from about 1 pg to 1 mg. In apreferred embodiment, the formulation contains tissue protectivecytokines that are non-erythropoietic.

In a further aspect of the invention, administration of tissueprotective cytokines was found to restore cognitive function in animalshaving undergone brain trauma. After a delay of either 5 days or 30days, administration of erythropoietin was still able to restorefunction as compared to sham-treated animals, indicating the ability ofan erythropoietin to regenerate or restore brain activity. Thus, theinvention is also directed to the use of erythropoietin and/or tissueprotective cytokines for the preparation of a pharmaceutical compositionfor the treatment of brain trauma and other cognitive dysfunctions,including treatment well after the injury (e.g. three days, five days, aweek, a month, or longer). The invention is also directed to a methodfor the treatment of cognitive dysfunction following injury byadministering an effective amount of erythropoietin and/or tissueprotective cytokines Any erythropoietin and/or tissue protectivecytokine as described herein may be used for this aspect of theinvention.

Furthermore, this restorative aspect of the invention is directed to theuse of any erythropoietins and/or tissue protective cytokines herein forthe preparation of a pharmaceutical composition for the restoration ofcellular, tissue or organ dysfunction, wherein treatment is initiatedafter, and well after, the initial insult responsible for thedysfunction. Moreover, treatment using erythropoietin and/or tissueprotective cytokines of the invention can span the course of the diseaseor condition during the acute phase as well as a chronic phase.

In the instance wherein an erythropoietin of the invention haserythropoietic activity, in a preferred embodiment, erythropoietin maybe administered systemically at a dosage between about 1 μg and about100 μg/kg body weight, preferably about 5-50 μg/kg-body weight, mostpreferably about 10-30 μg/kg-body weight, per administration. Thiseffective dose should be sufficient to achieve serum levels oferythropoietin greater than about 10,000, 15,000, or 20,000 mU/ml (80,120, or 160 ng/ml) of serum after erythropoietin administration. Suchserum levels may be achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10hours post-administration. Such dosages may be repeated as necessary.For example, administration may be repeated daily, as long as clinicallynecessary, or after an appropriate interval, e.g., every 1 to 12 weeks,preferably, every 1 to 3 weeks. In one embodiment, the effective amountof erythropoietin and a pharmaceutically acceptable carrier may bepackaged in a single dose vial or other container. In anotherembodiment, the tissue protective cytokines, which are capable ofexerting the activities described herein but not causing an increase inhemoglobin concentration or hematocrit, are used. Such tissue protectivecytokines are preferred in instances wherein the methods of the presentinvention are intended to be provided chronically. In anotherembodiment, an erythropoietin is given at a dose greater than thatnecessary to maximally stimulate erythropoiesis. As noted above, atissue protective cytokine of the invention does not necessarily haveerythropoietic activity, and therefore the above dosages expressed inhematopoietic units are merely exemplary for erythropoietins that areerythropoietic; hereinabove weight equivalents for dosages are providedwhich are applicable to tissue protective cytokines.

The present invention is further directed to a method for facilitatingthe transport of a molecule across an endothelial cell barrier in amammal by administering a composition which comprises the particularmolecule in association with an erythropoietin or tissue protectivecytokine as described hereinabove. As described above, tight junctionsbetween endothelial cells in certain organs in the body create a barrierto the entry of certain molecules. For treatment of various conditionswithin the barriered organ, means for facilitating passage ofpharmaceutical agents is desired. Erythropoietin or tissue protectivecytokines of the invention are useful as carriers for delivering othermolecules across the blood-brain and other similar barriers. Acomposition comprising a molecule desirous of crossing the barrier witherythropoietin or a tissue protective cytokine is prepared andperipheral administration of the composition results in the transcytosisof the composition across the barrier. The association between themolecule to be transported across the barrier and the erythropoietin ortissue protective cytokine may be a labile covalent bond, in which casethe molecule is released from association with the erythropoietin ortissue protective cytokine after crossing the barrier. If the desiredpharmacological activity of the molecule is maintained or unaffected byassociation with erythropoietin and or tissue protective cytokine, sucha complex can be administered.

The skilled artisan will be aware of various means for associatingmolecules with erythropoietin or a tissue protective cytokine of theinvention and the other agents described above, by covalent,non-covalent, and other means. Furthermore, evaluation of the efficacyof the composition can be readily determined in an experimental system.Association of molecules with erythropoietin or a tissue protectivecytokine may be achieved by any number of means, including labile,covalent binding, cross-linking, etc. Biotin/avidin interactions may beemployed; for example, a biotinylated erythropoietin of the inventionmay be complexed with a labile conjugate of avidin and a moleculedesirably transported. As mentioned above, a hybrid molecule may beprepared by recombinant or synthetic means, for example, a fusion orchimeric polypeptide which includes both the domain of the molecule withdesired pharmacological activity and the domain responsible forerythropoietin receptor activity modulation. Protease cleavage sites maybe included in the molecule.

A molecule may be conjugated to erythropoietin or a tissue protectivecytokine of the invention through a polyfunctional molecule, i.e., apolyfunctional crosslinker. As used herein, the term “polyfunctionalmolecule” encompasses molecules having one functional group that canreact more than one time in succession, such as formaldehyde, as well asmolecules with more than one reactive group. As used herein, the term“reactive group” refers to a functional group on the crosslinker thatreacts with a functional group on a molecule (e.g., peptide, protein,carbohydrate, nucleic acid, particularly a hormone, antibiotic, oranti-cancer agent to be delivered across an endothelial cell barrier) soas to form a covalent bond between the cross-linker and that molecule.The term “functional group” retains its standard meaning in organicchemistry. The polyfunctional molecules that can be used are preferablybiocompatible linkers, i.e., they are noncarcinogenic, nontoxic, andsubstantially non-immunogenic in vivo. Polyfunctional cross-linkers suchas those known in the art and described herein can be readily tested inanimal models to determine their biocompatibility. The polyfunctionalmolecule is preferably bifunctional. As used herein, the term“bifunctional molecule” refers to a molecule with two reactive groups.The bifunctional molecule may be heterobifunctional or homobifunctional.A heterobifunctional cross-linker allows for vectorial conjugation. Itis particularly preferred for the polyfunctional molecule to besufficiently soluble in water for the cross-linking reactions to occurin aqueous solutions such as in aqueous solutions buffered at pH 6 to 8,and for the resulting conjugate to remain water soluble for moreeffective bio-distribution. Typically, the polyfunctional moleculecovalently bonds with an amino or a sulfhydryl functional group.However, polyfunctional molecules reactive with other functional groups,such as carboxylic acids or hydroxyl groups, are contemplated in thepresent invention.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., Science 223, 1304-1306 (1984). Homobifunctional molecules having atleast two active ester units include esters of dicarboxylic acids andN-hydroxysuccinimide. Some examples of such N-succinimidyl estersinclude disuccinimidyl suberate and dithio-bis-(succinimidylpropionate), and their soluble bis-sulfonic acid and bis-sulfonate saltssuch as their sodium and potassium salts. These homobifunctionalreagents are available from Pierce, Rockford, Ill.

The heterobifunctional molecules have at least two different reactivegroups. The reactive groups react with different functional groups,e.g., present on the erythropoietin and the molecule. These twodifferent functional groups that react with the reactive group on theheterobifunctional cross-linker are usually an amino group, e.g., theepsilon amino group of lysine; a sulfhydryl group, e.g., the thiol groupof cysteine; a carboxylic acid, e.g., the carboxylate on aspartic acid;or a hydroxyl group, e.g., the hydroxyl group on serine.

Of course, certain of the various tissue protective cytokines of theinvention, and erythropoietin, may not have suitable reactive groupsavailable for use with certain cross-linking agent; however, one ofskill in the art will be amply aware of the choice of cross-linkingagents based on the available groups for cross-linking in erythropoietinor tissue protective cytokines of the invention.

When a reactive group of a heterobifunctional molecule forms a covalentbond with an amino group, the covalent bond will usually be an amido orimido bond. The reactive group that forms a covalent bond with an aminogroup may, for example, be an activated carboxylate group, ahalocarbonyl group, or an ester group. The preferred halocarbonyl groupis a chlorocarbonyl group. The ester groups are preferably reactiveester groups such as, for example, an N-hydroxy-succinimide ester group.

The other functional group typically is either a thiol group, a groupcapable of being converted into a thiol group, or a group that forms acovalent bond with a thiol group. The covalent bond will usually be athioether bond or a disulfide. The reactive group that forms a covalentbond with a thiol group may, for example, be a double bond that reactswith thiol groups or an activated disulfide. A reactive group containinga double bond capable of reacting with a thiol group is the maleimidogroup, although others, such as acrylonitrile, are also possible. Areactive disulfide group may, for example, be a 2-pyridyldithio group ora 5,5′-dithio-bis-(2-nitrobenzoic acid) group. Some examples ofheterobifunctional reagents containing reactive disulfide bonds includeN-succinimidyl 3-(2-pyridyl-dithio) propionate (Carlsson, et al., 1978,Biochem J., 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio) propionate is preferred. Someexamples of heterobifunctional reagents comprising reactive groupshaving a double bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl 3-(maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl) butyrate,sulfosuccinimidyl 4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce Chemical Co., Rockford, Ill. USA.

The need for the above-described conjugated to be reversible or labilemay be readily determined by the skilled artisan. A conjugate may betested in vitro for both the erythropoietin, and for the desirablepharmacological activity. If the conjugate retains both properties, itssuitability may then be tested in vivo. If the conjugated moleculerequires separation from erythropoietin or the tissue protectivecytokine for activity, a labile bond or reversible association witherythropoietin or the tissue protective cytokine will be preferable. Thelability characteristics may also be tested using standard in vitroprocedures before in vivo testing.

Additional information regarding how to make and use these as well asother polyfunctional reagents may be obtained from the followingpublications or others available in the art:

-   1. Carlsson, J. et al., 1978, Biochem. J. 173:723-737.-   2. Cumber, J. A. et al., 1985, Methods in Enzymology 112:207-224.-   3. Jue, R. et al., 1978, Biochem 17:5399-5405.-   4. Sun, T. T. et al., 1974, Biochem. 13:2334-2340.-   5. Blattler, W. A. et al., 1985, Biochem. 24:1517-152.-   6. Liu, F. T. et al., 1979, Biochem. 18:690-697.-   7. Youle, R. J. and Neville, D. M. Jr., 1980, Proc. Natl. Acad. Sci.    U.S.A. 77:5483-5486.-   8. Lerner, R. A. et al., 1981, Proc. Natl. Acad. Sci. U.S.A.    78:3403-3407.-   9. Jung, S. M. and Moroi, M., 1983, Biochem. Biophys. Acta 761:162.-   10. Caulfield, M. P. et al., 1984, Biochem. 81:7772-7776.-   11. Staros, J. V., 1982, Biochem. 21:3950-3955.-   12. Yoshitake, S. et al., 1979, Eur. J. Biochem. 101:395-399.-   13. Yoshitake, S. et al., 1982, J. Biochem. 92:1413-1424.-   14. Pilch, P. F. and Czech, M. P., 1979, J. Biol. Chem.    254:3375-3381.-   15. Novick, D. et al., 1987, J. Biol. Chem. 262:8483-8487.-   16. Lomant, A. J. and Fairbanks, G., 1976, J. Mol. Biol.    104:243-261.-   17. Hamada, H. and Tsuruo, T., 1987, Anal. Biochem. 160:483-488.-   18. Hashida, S. et al., 1984, J. Applied Biochem. 6:56-63.

Additionally, methods of cross-linking are reviewed by Means and Feeney,1990, Bioconjugate Chem. 1:2-12.

Barriers which are crossed by the above-described methods andcompositions of the present invention include but are not limited to theblood-brain barrier, the blood-eye barrier, the blood-testes barrier,the blood-ovary barrier, and the blood-uterus barrier.

Candidate molecules for transport across an endothelial cell barrierinclude, for example, hormones, such as growth hormone, neurotrophicfactors, antibiotics, antivirals, or antifungals such as those normallyexcluded from the brain and other barriered organs, peptideradiopharmaceuticals, antisense drugs, antibodies and antivirals againstbiologically-active agents, pharmaceuticals, and anti-cancer agents.Non-limiting examples of such molecules include hormones such as growthhormone, nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growthfactor (bFGF), transforming growth factor β1 (TGFβ1), transforminggrowth factor β2 (TGFβ2), transforming growth factor β3 (TGFβ3),interleukin 1, interleukin 2, interleukin 3, and interleukin 6, AZT,antibodies against tumor necrosis factor, and immunosuppressive agentssuch as cyclosporin. Additionally, dyes or markers may be attached toerythropoietin or one of the tissue protective cytokines of the presentinvention in order to visualize cells, tissues, or organs within thebrain and other barriered organs for diagnostic purposes. As an example,a marker used to visualize plaque within the brain could be attached toerythropoietin or a tissue protective cytokine in order to determine theprogression of Alzheimer's disease within a patient.

The present invention is also directed to a composition comprising amolecule to be transported via transcytosis across an endothelial celltight junction barrier and an erythropoietin or tissue protectivecytokine as described above. The invention is further directed to theuse of a conjugate between a molecule and an erythropoietin or a tissueprotective cytokine as described above for the preparation of apharmaceutical composition for the delivery of the molecule across abarrier as described above.

In the following examples, various animal models and in-vitro tests ofneuroprotection and transcytosis are provided to demonstrate theeffectiveness of the tissue protective cytokines of the invention. Suchmodels include in vitro models using P-19 cells to determine theneuroprotective affects of the tissue protective cytokines, and in-vivowater intoxication model in mice to determine the in vivoneuroprotective affects of the tissue protective cytokines of thepresent invention. For transcytosis, model proteins conjugated to theerythropoietins of the invention are evaluated for transport into thebrain following parenteral administration. These tests in in-vitromodels and animal models are predictive of the efficacy of the presentcompounds in other mammalian species including humans. Additionally,Example 1, demonstrates that the human brain has an abundance oferythropoietin receptors that provide the mechanism for the transcytosisof erythropoietin as well as tissue protective cytokines.

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. The following examples are presented in order to more fullyillustrate the preferred embodiments of the invention. They should in noway be construed, however, as limiting the broad scope of the invention.

Example 1 Distribution of Erythropoietin Receptor in Human Brain

Normal human brains removed during surgical procedures (e.g., to providetumor-free margins in tumor resections) were immediately fixed in 5%acrolein in 0.1 M phosphate buffer (pH 7.4) for 3 h. Sections were cutwith a vibrating microtome at 40 micrometer thickness.Immunohistochemical staining was performed using free-floating sectionsand the indirect antibody peroxidase-antiperoxidase method using a 1:500dilution of erythropoietin receptor antiserum (obtained from Santa CruzBiotechnology). Endogenous peroxidase activity was quenched bypretreatment of tissue sections with hydrogen peroxide (3% in methanolfor 30 min). Tissue controls were also carried out by primary antibodyomission and by using the appropriate blocking peptide (from Santa CruzBiotech.) to confirm that staining was specific for erythropoietin (EPO)receptor.

FIG. 1 shows that capillaries of the human brain express very highlevels of EPO receptor, as determined by immunohistochemistry usingspecific anti-EPO receptor antibodies. This provides a mechanism wherebyEPO is able to penetrate into the brain from the systemic circulation,in spite of the blood brain barrier.

FIG. 2 shows the EPO receptor is densely localized within and aroundcapillaries forming the blood brain barrier in the human brain.

A similar protocol as for FIGS. 1 & 2 was performed for FIG. 3, exceptthat 10 micrometer sections were cut from paraffin, the embeddedsections fixed by immersion in 4% paraformaldehyde. FIG. 3 shows thatthere is a high density of EPO receptor at the luminal and anti-luminalsurfaces of human brain capillaries, forming the anatomical basis fortransport of EPO from the circulation into the brain.

FIG. 4 was obtained following a similar protocol as in FIG. 3 exceptthat the tissue was sectioned on an ultramicrotome for electronmicroscopy and the secondary antibody was labeled with colloidal goldparticles. This figure shows that EPO receptor is found upon theendothelial surface (*), within cytoplasmic vesicles (arrows) and uponglial endfeet (+) in human brain, providing the anatomical basis fortransport of EPO from within the circulation into the brain.

Example 2 Tissue Protective Cytokines

Tissue protective cytokines desirable for the uses described herein maybe generated by guanidination, carbamylation, amidination,trinitrophenylation, acetylation, succinylation, nitration, ormodification of arginine or lysine residues or carboxyl groups, amongother procedures as mentioned herein above, of erythropoietin. Thesemodifications produce tissue protective cytokines that maintain theiractivities for specific organs and tissues but not for others, such aserythrocytes. When erythropoietin is subjected to the above reactions,it has been found that in general the resultant molecule lacks bothin-vivo and in-vitro erythropoietic activity (e.g., Satake et al; 1990,Biochim. Biophys. Acta 1038:125-9). Some examples of the preparation oftissue protective cytokines are described below. Although the examplesbelow use erythropoietin as the starting material, one of ordinary skillin the art would recognize that erythropoietin derivatives such asdesialylated, guanidinated, carbamylated, amidinated,trinitrophenylated, acetylated, succinylated, and nitratederythropoietin can be used as well.

A. Production of Tissue Protective Cytokines by DesialylatingErythropoietin

Erythropoietin may be desialylated by the following exemplary procedure.Sialidase (isolated from Streptococcus sp 6646K.) is obtained fromSEIKAGAKU AMERICA (Code No. 120050). Erythropoietin is subjected todesialylation by sialidase (0.05 U/mg EPO) at 37 C for 3 h. The reactionmixture is desalted and concentrated using an Ultrafree CentrifugalFilter Unit. The sample is then applied to an ion exchange column inAKTAprime™ system. The protein is eluted with selected buffers. Theeluted fractions containing a significant amount of protein are thensubjected to IEF gel analysis. The fractions containing only the top twobands (migrating at pI ˜8.5 and ˜7.9 on IEF gel) are pooled. The proteincontent of the pooled fractions was determined and 1/9 volumes of 10×salt solution (1 M NaCl, 0.2 M sodium citrate, 3 mM citric acid) wasadded. The sialic acid content of the solution was then determined. Nosignificant sialic content should be detected.

Asialoerythropoietin and phenylglyoxalerythropoietin were as effectiveas native erythropoietin for neural cells in vitro as shown in FIGS.5-6. In-vitro testing was carried out using neural-like embryonalcarcinoma cells (P19) that undergo apoptosis upon the withdrawal ofserum. Twenty-four hours before the removal of serum, 1-1000 ng/ml oferythropoietin or a modified erythropoietin was added to the cultures.The following day the medium was removed, the cells washed with fresh,non-serum containing medium, and medium containing the test substance(no serum) added back to the cultures for and additional 48 hours. Todetermine the number of viable cells, a tetrazolium reduction assay wasperformed (CellTiter 96; Promega, Inc.). As FIG. 5-6 illustrate,asialoerythropoietin appears to be of equal potency to erythropoietinitself in preventing cell death.

Retention of neuroprotective activity in vivo was confirmed using a ratfocal ischemia model in which a reversible lesion in the territory ofthe middle cerebral artery is performed as described previously (Brineset al., 2000, Proc. Nat. Acad. Sci. U.S.A. 97:10526-31). Adult maleSprague-Dawley rats were administered asialoerythropoietin orerythropoietin (5000 U (40 μg)/kgBW intraperitoneally) or vehicle at theonset of the arterial occlusion. Twenty-four hours later, the animalswere sacrificed and their brains removed for study. Serial sections werecut and stained with tetrazolium salts to identify living regions of thebrain. As shown in FIG. 7, asialoerythropoietin was as effective asnative erythropoietin in providing neuroprotection from 1 hour ofischemia. FIG. 8 shows the results of another focal ischemia model inwhich a comparative dose response was performed with erythropoietin andasialoerythropoietin. At the lowest dose of 250 U (2 μg)/kg,asialoerythropoietin afforded protection whereas unmodifiederythropoietin did not.

B. Preparation of Tissue Protective Cytokines by CarbamylatingErythropoietin

Native erythropoietin may be used to prepare the respective carbamylatedmolecules, in accordance with the following procedure, as described inJin Zeng (1991). Lysine modification of metallothionein by carbamylationand guanidination. Methods in Enzymology 205: 433-437. First, potassiumcyanate was recrystallized. A 1 M Borate buffer (pH 8.8) was prepared.An erythropoietin solution was mixed with an equal volume of the boratebuffer. Potassium cyanate was added directly to the reaction tube to afinal concentration of 0.5 M. The solution was mixed well and incubatedat 37 C for 6 h. The solution was then dialyzed thoroughly usingdistilled water. The product was removed from the dialysis tubing andcollected into a fresh tube. The volume was measured and 1/9 volume of10× salt solution (1 M NaCl, 0.2 M sodium citrate, 3 mM citric acid) isadded to the solution. The protein content is determined and the productrecovery rate is calculated. The products were analyzed by IEF gelfollowed by an in vitro test with TF-1 cells.

C. Preparation of Tissue Protective Cytokines by SuccinylatingErythropoietin

Native erythropoietin may be used to prepare the respective succinylatedmolecules, in accordance with the following procedure, as described inAlcalde et al. (2001). Succinylation of cyclodextrin glycosyltransferasefrom Thermoanaerobacter sp. 501 enhances its transferase activity usingstarch as donor. J. Biotechnology 86: 71-80. Erythropoietin (100 ug) in0.5 M NaHCO3 (pH 8.0) was incubated with a 15 molar excess of succinicanhydride at 15 C for 1 hour. The reaction was stopped by dialysisagainst distilled water.

Another method for succinylating erythropoietin is to dissolve succinicanhydride in dry acetone at 27 mg/ml. The reaction is performed in aneppendorf tube in 10 mM sodium phosphate buffer (pH 8.0). Erythropoietinand 50-fold molar of succinic anhydride are added to the tube. Thesolution is mixed well and the tube is rotated at 4 C for 1 h. Thereaction is stopped by dialysis against 10 mM sodium phosphate buffer,using a Dialysis cassette (Slide-A-Laze 7K, Pierce 66373). The productis removed from the dialysis cassette and collected into a fresh tube.The volume is measured and 1/9 volume of 10× salt solution (1 M NaCl,0.2 M sodium citrate, 3 mM citric acid) is added. Determine the proteincontent and calculate the product recovery rate. The products wereanalyzed by IEF gel followed by an in vitro test with TF-1 cells.

D. Preparation Tissue Protective Cytokine by Acetylating Erythropoietin

Native erythropoietin may be used to prepare the respective acetylatedmolecules, in accordance with the following procedure, as described inSatake et al (1990). Chemical modification of erythropoietin: anincrease in in-vitro activity by guanidination. Biochimica et BiophysicaActa. 1038: 125-129.

The reaction was performed in an eppendorf tube in 80 mM sodiumphosphate buffer (pH 7.2). Erythropoietin and equal molar of aceticanhydride were added to the tube. After mixing well, the solution wasincubated on ice for 1 h. The reaction was stopped by dialysis againstwater, using a Dialysis cassette (Slide-A-Laze 7K, Pierce 66373). Theproduct was removed from the dialysis cassette and collected into afresh tube. After measuring the volume of product, 1/9 volume of 10×salt solution (1 M NaCl, 0.2 M sodium citrate, 3 mM citric acid) wasadded. The protein content is determined, and the product recovery ratewas calculated. The product was analyzed by IEF gel followed by an invitro test with TF-1 cells.

E. Preparation of Tissue Protective Cytokine by CarboxymethylatingLysine of Erythropoietin

Native erythropoietin may be used to prepare the respectiveN^(ε)-(carboxymethyl)lysine (CML) modified molecules in which one ormore lysyl residues of the erythropoietin are modified, in accordancewith the following procedure, as described in Akhtar et al (1999)Conformational study of N^(ε)-(carboxymethyl)lysine adducts ofrecombinant a-crystallins. Current Eye Research, 18: 270-276.

Glyoxylic acid (200 mM) and NaBH₃CN (120 mM) were prepared in sodiumphosphate buffer (50 mM, pH 7.5). In an eppendorf tube, erythropoietinwas added (in phosphate buffer). The lysine equivalent in the solution(about 8 lysine residues/mol) was then calculated. Next, 3-times greaterNaBH₃CN and 5 or 10-times greater glyoxylic acid was added to the tube.Each tube was vortexed and incubated at 37 C for 5 h. The samples weredialated against phosphate buffer overnight at 4 C. The volume of eachproduct was measured after dialysis. The protein concentration wasdetermined, and the product recovery rate was calculated. The productwas analyzed by IEF gel followed by an in vitro test with TF-1 cells.

F. Preparation of Tissue Protective Cytokine by IodinatingErythropoietin

Native erythropoietin may be used to prepare the respective iodinatedmolecules, in accordance with the following procedure, as described ininstruction provided by Pierce Chemical Company (Rockford, Ill.) forIODO-Gen Pre-Coated Iodination Tubes (product #28601).

First, 0.1 M of NaI was prepared, and iodination was performed in anIODO-Gen Pre-Coated Iodination Tube (Pierce, 28601), with a totalreaction volume of 0.1 ml/tube in sodium phosphate buffer (40 mM, pH7.4). The protein substrate (erythropoietin) was mixed with sodiumphosphate buffer and then transferred to an IODO-Gen Pre-CoatedIodination Tube. NaI was added to a final concentration of 1-2 mM,making the molar ration of NaI/protein as 14-20. The solution was thenmixed well and incubated at room temperature for 15 min with gentleagitation. The reaction was stopped by removing the reaction mixture andadding to it a tube containing 3.9 ml of sodium buffer (i.e., a 40-folddilution). The product was concentrated by a pre-wet Ultrafreecentrifugal filter unit. The volume of concentrate was measured and 1/9volume of 10× salt solution (1 M NaCl, 0.2 M sodium citrate, 3 mM citricacid) was added. The protein concentration was determined, and theproduct recovery was then calculated. The products were analyzed by IEFgel followed by an in vitro test with TF-1 cells.

2. Another method for iodinating erythropoietin involves incubating oneIodo Bead (Pierce, Rockford, Il) in 100 ul PBS (20 mM sodium phosphate,0.15M NaCl, pH7.5) containing 1 mCi free Na¹²⁵I for 5 minutes.Erythropoietin (100 ug) in 100 ul PBS was then added to the mixture.After a ten minute incubation period at room temperature, the reactionwas stopped by removing the 200 ul solution from the reaction vessel(leaving the iodo bead behind). The excess iodine was then removed bygel filtration on a Centricon 10 column. As shown in FIG. 9,iodo-erythropoietin produced in this manner is efficacious in protectingP19 cells from serum withdrawal.

3. Erythropoietin may also be iodinated using Chloramine T.Erythropoietin (100 ug) in 100 ul PBS was added to 500 uCi Na¹²⁵I, andthe mixture was then mixed together in an eppendorf tube. 25 ulchloramines T (2 mg/ml) were then added and the mixture was incubatedfor 1 minute at room temperature. 50 ul of Chloramine T stop buffer (2.4mg/ml sodium metabisulfite, 10 mg/ml tyrosine, 10% glycerol, 0.1% xylenein PBS was then added. The iodotyrosine and iodinated erythropoietinwere then separated by gel filtration on a Centricon 10 column.

G. Preparation of Tissue Protective Cytokine by BiotinylatingErythropoietin

1. In “Biotinylated recombinant human erythropoietins: Bioactivity andUtility as a receptor ligand” by Wojchowski et al. Blood, 1989,74(3):952-8, the authors use three different methods of biotinylatingerythropoietin. Biotin is added to (1) the sialic acid moieties (2)carboxylate groups and (3) amino groups. The authors use a mouse spleencell proliferation assay to demonstrate that (1) the addition of biotinto the sialic acid moieties does not inactivate the biological activityof erythropoietin (2) the addition of biotin to carboxylate groups ledto substantial biological inactivation of erythropoietin (3) theaddition of biotin to amino groups resulted in complete biologicalinactivation of erythropoietin. These methods and modifications arefully embraced herein. FIG. 10 shows the activity of biotinylatederythropoietin and asialoerythropoietin in the serum-starved P19 assay.

2. Additionally, native erythropoietin may be used to prepare therespective biotinylated molecules, in accordance with the followingprocedure, as described in instruction provided by Pierce ChemicalCompany (Rockford, Ill.) for EZ-Link NHS-LC-Biotin (product #21336).

Immediately before the reaction, EZ-Link NHS-LC-Biotin (pierce, 21336)in DMSO at 2 mg/ml was dissolved. The reaction was performed in a tube(17×100 mm) with total volume of 1 ml containing 50 mM sodiumbicarbonate (pH 8.3). Erythropoietin and <10% of EZ-Link NHS-LC-Biotinwere added to generate a solution with a molar ratio of Biotin/proteinat ˜20. The solution was mixed well and incubated on ice for 2 h. Thesolution was desalted and concentrated using an Ultrafree centrifugalfilter unit. The product was then collected into a fresh tube. Thevolume of the product was measured, and 1/9 volume of 10× salt solution(1 M NaCl, 0.2 M sodium citrate, 3 mM citric acid) was added to theproduct. The protein content of the product was determined and theproduct recovery rate was calculated. The products were analyzed by IEFgel followed by an in vitro test with TF-1 cells.

3. The free amino groups of erythropoietin can also be biotinylatedusing the following method. First, 0.2 mg D-biotinoyl-e-aminocaproicacid-N-hydroxysuccinimide ester (Boehringer Mannheim #1418165) wasdissolved in 100 ul DMSO. This solution was then combined with 400 ulPBS containing approximately 0.2 mg erythropoietin in a foil coveredtube. After incubating this solution for 4 hours at room temperature,the unreacted biotin was separated by gel filtration on a Centricon 10column.

It is contemplated that several of these modifications may be performedon erythropoietin or an erythropoietin derivative in order to arrive ata tissue protective cytokine. For example erythropoietin can bedesialylated in accordance with the procedure listed above at Example2(A) and carbamylated in accordance with the procedure listed above atExample 2(B) to generate an asialo carbamoylerythropoietin.

Example 3 Preparation of Tissue Protective Cytokines by Other Methods

1. Trinitrophenylation: erythropoietin (100 ug) was modified with2,4,6-trinitrobenzenesulfonate as described in Plapp et al (“Activity ofbovine pancreatic deoxyribonuclease A with modified amino groups” 1971,J. Biol. Chem. 246, 939-845)

2. Arginine modifications: erythropoietin was modified with 2,3butanedione as described in Riordan (“Functional arginyl residues incarboxypeptidase A. Modification with butanedione” Riordan J F,Biochemistry 1973, 12(20): 3915-3923).

3. Erythropoietin was modified with cylcohexanone as in Patthy et al(“Identification of functional arginine residues in ribonuclease A andlysozyme” Patthy, L, Smith E L, J. Biol. Chem 1975 250(2): 565-9).

4. Erythropoietin was modified with phenylglyoxal as described in Werberet al. (“Proceedings: Carboxypeptidase B: modification of functionalarginyl residues” Werber, M M, Sokolovsky M Isr J Med Sci 1975 11(11):1169-70). The phenylglyoxal-modified erythropoietin was tested using theneural-like P19 cell assay described above. As FIG. 11 illustrates, thischemically-modified erythropoietin fully retains its neuroprotectiveeffects.

5. Tyrosine modifications: erythropoietin (100 ug) was incubated withtetranitromethane as previously described in Nestler et al “Stimulationof rat ovarian cell steroidogenesis by high density lipoproteinsmodified with tetranitromethane” Nestler J E, Chacko G K, Strauss J F3rd. J Biol Chem 1985 Jun. 25; 260(12):7316-21).

6. Glutamic acid (and aspartic acid) modifications: In order to modifycarboxyl groups, erythropoietin (100 ug) was incubated with 0.02 M EDCin 1M glycinamide at pH 4.5 at room temperature for 60 minutes asdescribed in Carraway et al “Carboxyl group modification in chymotrypsinand chymotrypsinogen.” Carraway K L, Spoerl P, Koshland D E Jr. J MolBiol 1969 May 28; 42(1):133-7.

7. Tryptophan residue modifications: erythropoietin (100 ug) wasincubated with 20 uM n-bromosuccinimide in 20 mM potassium phosphatebuffer (pH 6.5) at room temperature as described in Ali et al., J BiolChem. 1995 Mar. 3; 270(9):4570-4. The number of oxidized tryptophanresidues was determined by the method described in Korotchkina(Korotchkina, L G et al Protein Expr Purif. 1995 February; 6(1):79-90).

8. Removal of amino groups: In order to remove amino groups oferythropoietin (100 ug) was incubated with in PBS (pH 7.4) containing 20mM ninhydrin (Pierce Chemical, Rockford, Il), at 37 C for two hours asin Kokkini et al (Kokkini, G., et al “Modification of hemoglobin byninhydrin” Blood, Vol. 556, No 4 1980: 701-705). Reduction of theresulting aldehyde was accomplished by reacting the product with sodiumborohydride or lithium aluminum hydride. Specifically, erythropoietin(100 ug) was incubated with 0.1M sodium borohydride in PBS for 30minutes at room temperature. The reduction was terminated by cooling thesamples on ice for 10 minutes and dialyzing it against PBS, three times,overnight. (Kokkini, G., Blood, Vol. 556, No 4 1980: 701-705). Reductionusing lithium aluminum hydride was accomplished by incubatingerythropoietin (100 ug) with 0.1M lithium aluminum hydride in PBS for 30minutes at room temperature. The reduction was terminated by cooling thesamples on ice for 10 minutes and dialyzing the samples against PBS,three times, overnight.

9. Disulfide reduction and stabilization: erythropoietin (100 ug) wasincubated with 500 mM DTT for 15 minutes at 60 C. 20 mM iodoacetamide inwater was then added to the mixture and incubated for 25 minutes, atroom temperature in the dark.

10. Limited proteolysis: Erythropoietin can be subjected to a limitedchemical proteolysis that targets specific residues. Erythropoietin wasreacted with 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolenine whichcleaves specifically after tryptophan residues in a 50 times excess in50% acetic acid for 48 hours in the dark at room temperature in tubescapped under nitrogen pressure. The reaction was terminated by quenchingwith tryptophan and desalting.

As noted above, erythropoietin or asialoerythropoietin may be modified,yet multiple modifications as well as additional modifications of theerythropoietin molecule may also be performed without deviating from thespirit of the present invention. Any of the foregoing examples may becarried out with partially desialylated erythropoietin, which may beprepared as described below. For example, any of the aforementionedmodified erythropoietins may be modified at one or more arginineresidues by using, for example, phenylglyoxal according to the protocolof Takahashi (1977, J. Biochem. 81:395-402), which may be carried outfor variable lengths of time ranging from 0.5 to 3 hrs at roomtemperature. The reaction was terminated by dialyzing the reactionmixture against water. Use of such modified forms of erythropoietin isfully embraced herein.

Example 4 Tissue Protective Cytokines have Neuro Protective Effect

The neuroprotective affects of the tissue protective cytokines of thepresent invention was evaluated using a water intoxication assay inaccordance with Manley et al., 2000, Aquaporin-4 deletion in micereduces brain edema after acute water intoxication and ischemic stroke,Nat Med 2000 February; 6(2):159-63. Female C3H/HEN mice were used. Themice were given 20% of their body weight as water IP with 400 ng/kg bwDDAVP (desmopressin). The mice were administered erythropoietin (A) or atissue protective cytokine: asialoerythropoietin (B), carbamylatedasialoerythropoietin (C), succinylated asialoerythropoietin (D),acetylated asialoerythropoietin (E), iodinated asialoerythropoietin (F),carboxymethylated asialoerythropoietin (G), carbamylated erythropoietin(H), acetylated erythropoietin (I), iodinated erythropoietin (J), orN^(E)-carboxy methyl erythropoietin (K). The mice were given a 100microgram/kg dose of erythropoietin or tissue protective cytokineintraperitoneally 24 hrs before administration of the water and again atthe time of the water administration. A modified scale from Manley etal. was used to evaluate the mice. The modified scale is as listedbelow:

1. Explores cage/table Yes 0 No 1 2. Visually tracks objects Yes 0 No 13. Whisker movement Present 0 Absent 1 4. Leg-tail movements Normal 0Stiff 1 Paralyzed 2 5. Pain withdrawal (toe pinch) Yes 0 No 1 6.Coordination of movement Normal 0 Abnormal 1 7. Stops at edge of tableYes 0 No 1 Total score possible: 8

The mice were scored at the following time points: 15, 30, 45, 60, 75,90, 120, 150, 180 minutes. Score as plotted is the area under the entiretime curve, as percent of animals that had received saline only. FIG. 12shows the scores of the mice who received erythropoietin or one of thetissue protective cytokines of the present invention as a percentage ofthe score obtained by the control mice. The mice who received the tissueprotective cytokines of the present invention exhibited lessneurological damage and therefore scored better on the modified scale.FIG. 12 shows that the tissue protective cytokines of the presentinvention protect neuronal tissue.

Example 5 Erythropoietin Crosses the Blood-Cerebrospinal Fluid TightBarrier

Adult male Sprague-Dawley rats were anesthetized and administeredrecombinant human erythropoietin intraperitoneally. Cerebrospinal fluidwas sampled from the cisterna magna at 30 minute intervals up to 4 hrsand the erythropoietin concentration determined using a sensitive andspecific enzyme-linked immunoassay. As illustrated in FIG. 13, thebaseline erythropoietin concentration in CSF is 8 mU/ml. After a delayof several hours, the levels of erythropoietin measured in the CSF beginto rise and by 2.5 hours and later are significantly different from thebaseline concentration at the p<0.01 level. The peak level of about 100mU/ml is within the range known to exert protective effects in vitro(0.1 to 100 mU/ml). The time to peak occurs at about 3.5 hrs, which isdelayed significantly from the peak serum levels (less than 1 hr). Theresults of this experiment illustrate that significant levels oferythropoietin can be accomplished across a tight cellular junction bybolus parenteral administration of erythropoietin at appropriateconcentrations. One of ordinary skill in the art would recognize thatsimilar results would be expected from the tissue protective cytokinesof the present invention.

Example 6 Maintenance of Function in Heart Prepared for Transplantation

Wistar male rats weighing 300 to 330 g are given erythropoietin (5000U/kg body weight) or vehicle 24 h prior to removal of the heart for exvivo studies, done in accordance with the protocol of Delcayre et al.,1992, Amer. J. Physiol. 263:H1537-45. Animals are sacrificed withpentobarbital (0.3 mL), and intravenously heparinized (0.2 mL). Thehearts are initially allowed to equilibrate for 15 min. The leftventricular balloon is then inflated to a volume that gives anend-diastolic pressure of 8 mm Hg. A left ventricular pressure-volumecurve is constructed by incremental inflation of the balloon volume by0.02 ml aliquots. Zero volume is defined as the point at which the leftventricular end-diastolic pressure is zero. On completion of thepressure-volume curve, the left ventricular balloon is deflated to setend-diastolic pressure back to 8 mmHg and the control period is pursuedfor 15 min., after check of coronary flow. Then the heart is arrestedwith 50 mL Celsior+molecule to rest at 4° C. under a pressure of 60 cmH₂0. The heart is then removed and stored 5 hours at 4° C. in plasticcontainer filled with the same solution and surrounded with crushed ice.

On completion of storage, the heart is transferred to a Langendorffapparatus. The balloon catheter is re-inserted into the left ventricleand re-inflated to the same volume as during preischemic period. Theheart is re-perfused for at least 2 hours at 37° C. The re-perfusionpressure is set at 50 cm H₂0 for 15 min of re-flow and then back to 100cm H₂0 for the 2 next hours. Pacing (320 beats per minute) isre-instituted. Isovolumetric measurements of contractile indexes anddiastolic pressure are taken in triplicate at 25, 45, 60, 120 min ofreperfusion. At this time point pressure volume curves are performed andcoronary effluent during the 45 min reperfusion collected to measurecreatine kinase leakage. The two treatment groups are compared using anunpaired t-test, and a linear regression using the end-diastolicpressure data is used to design compliance curves. As shown in FIG. 14,significant improvement of left ventricular pressure developed occursafter treatment with erythropoietin, as well as improved volume-pressurecurve, decrease of left diastolic ventricular pressure and decrease ofcreatine kinase leakage. Similar results would be expected fromtreatment with the tissue protective cytokines of the present invention.

Example 7 Erythropoietin Protects Myocardium from Ischemic Injury

Adult male rats given recombinant human erythropoietin (5000 U (40μg)/kg body weight) 24 hrs previously are anesthetized and prepared forcoronary artery occlusion. An additional dose of erythropoietin is givenat the start of the procedure and the left main coronary artery occludedfor 30 minutes and then released. The same dose of erythropoietin isgiven daily for one week after treatment. The animals are then studiedfor cardiac function. As FIG. 15 illustrates, animals receiving a shaminjection (saline) demonstrated a large increase in the left enddiastolic pressure, indicative of a dilated, stiff heart secondary tomyocardial infarction. In contradistinction, animals receivingerythropoietin suffered no decrement in cardiac function, compared tosham operated controls (difference significant at the p<0.01 level).Similar results would be expected from treatment with the tissueprotective cytokines of the present invention.

Example 8 Protection of Retinal Ischemia by Peripherally-AdministeredErythropoietin

Retinal cells are very sensitive to ischemia such that many will dieafter 30 minutes of ischemic stress. Further, subacute or chronicischemia underlies the deterioration of vision which accompanies anumber of common human diseases, such as diabetes mellitus, glaucoma,and macular degeneration. At the present time there are no effectivetherapies to protect cells from ischemia. A tight endothelial barrierexists between the blood and the retina that excludes most largemolecules. To test whether peripherally-administered erythropoietin willprotect cells sensitive to ischemia, an acute, reversible glaucoma ratmodel was utilized as described by Rosenbaum et al. (1997; Vis. Res.37:3443-51). Specifically, saline was injected into the anterior chamberof the eye of adult male rats to a pressure above systemic arterialpressure and maintained for 60 minutes. Animals were administered salineor 5000 U (40 μg) erythropoietin/kg body weight intraperitoneally 24hours before the induction of ischemia, and continued as a daily dosefor 3 additional days. Electroretinography was performed on dark-adaptedrats 1 week after treatment. FIG. 16-17 illustrate that theadministration of erythropoietin is associated with good preservation ofthe electroretinogram (ERG) (Panel D), in contrast to animals treatedwith saline alone (Panel C), for which very little function remained.FIG. 16 compares the electroretinogram a- and b-wave amplitudes for theerythropoietin-treated and saline-treated groups, and shows significantprotection afforded by erythropoietin. Similar results are obtainablefrom treatment with the tissue protective cytokines of the presentinvention.

Example 9 Restorative Effects of Erythropoietin on Diminished CognitiveFunction Arising from Brain Injury

In a study to demonstrate the ability of erythropoietin to restorediminished cognitive function in mice after receiving brain trauma,female Balb/c mice were subject to blunt brain trauma as described inBrines et al. PNAS 2000, 97; 10295-10672 and five days later, dailyerythropoietin administration of 5000 U (40 μg)/kg-bw intraperitoneallywas begun. Twelve days after injury, animals were tested for cognitivefunction in the Morris water maze, with four trials per day. While bothtreated and untreated animals performed poorly in the test (with swimtimes of about 80 seconds out of a possible 90 seconds), FIG. 18 showsthat the erythropoietin-treated animals performed better (in thispresentation, a negative value is better). Even if the initiation oferythropoietin treatment is delayed until 30 days after trauma (FIG.19), restoration of cognitive function is also seen. Similar resultswould be expected from treatment with the tissue protective cytokines ofthe present invention.

Example 10 Kainate Model

In the kainate neurotoxicity model, asialoerythropoietin wasadministered according to the protocol of Brines et al. Proc. Nat. Acad.Sci. U.S.A. 2000, 97; 10295-10672 at a dose of 5000 U (40 μg)/kg-bwgiven intraperitoneally 24 hours before the administration of 25 mg/kgkainate is shown to be as effective as erythropoietin, as shown by timeto death (FIG. 20). Similar results are obtainable from treatment withthe tissue protective cytokines of the present invention.

Example 11 Spinal Cord Injury Models

1. Rat Spinal Cord Compression Testing Erythropoietin and TissueProtective Cytokines

Wistar rats (female) weighing 180-300 g were used in this study. Theanimals were fasted for 12 h before surgery, and were humanelyrestrained and anesthetized with an intraperitoneal injection ofthiopental sodium (40 mg/kg-bw). After infiltration of the skin(bupivacaine 0.25%), a complete single level (T-3) laminectomy wasperformed through a 2 cm incision with the aid of a dissectingmicroscope. Traumatic spinal cord injury was induced by the extraduralapplication of a temporary aneurysm clip exerting a 0.6 newton (65grams) closing force on the spinal cord for 1 minute. After removal ofthe clip, the skin incision was closed and the animals allowed torecover fully from anesthesia and returned to their cages. The rats weremonitored continuously with bladder palpation at least twice daily untilspontaneous voiding resumed.

40 animals were randomly divided into five groups. Animals in thecontrol group (I) (n=8) received normal saline (via intravenousinjection) immediately after the incision is closed. Group (II; n=8)received rhEPO @ 16 micrograms/kg-bw iv; group (III) received an asialotissue protective cytokine of the present invention(asialoerythropoietin) @ 16 micrograms/kg-bw iv, group (IV) received anasialo tissue protective cytokine @ 30 micrograms/kg-bw iv, and group(V) received an asialo tissue protective cytokine of the presentinvention (asialoerythropoietin) @ 30 micrograms/kg-bw; all as a singlebolus intravenous injection immediately after removal of the aneurysmclip.

Motor neurological function of the rats will be evaluated by use of thelocomotor rating scale of Basso et al. In this scale, animals areassigned a score ranging from 0 (no observable hindlimb movements) to 21(normal gait). The rats will be tested for functional deficits at 1, 12,24, 48, 72 hours and then at 1 week after injury by the same examinerwho is blind to the treatment each animal receives.

FIG. 21 is a graph demonstrating the locomotor ratings of the ratsrecovering from the spinal cord trauma over a period of thirty days. Ascan be seen from the graph, the rats that were given erythropoietin(group II) or tissue protective cytokines (groups III-V) recovered fromthe injury more readily and demonstrated better overall recovery fromthe injury than the control rats. Similar results would be expected fromthe therapeutic treatment with the tissue protective cytokines of thepresent invention.

2. Rabbit Spinal Cord Ischemia Testing Erythropoietin and a TissueProtective Cytokine.

36 New Zealand White rabbits (8-12 months old, male) weighing 1.5-2.5 kgwere used in this study. The animals were fasted for 12 hours andhumanely restrained. Anesthesia induction was via 3% halothane in 100%oxygen and maintained with 0.5-1.5% halothane in a mixture of 50% oxygenand 50% air. An intravenous catheter (22 gauge) was placed in the leftear vein. Ringers lactate was infused at a rate of 4 ml/kg body weight(bw) per hour during the surgical procedure. Preoperatively, cefazoline10 mg/kg-bw was administered intravenously for prophylaxis of infection.The animals were placed in the right lateral decubitus position, theskin prepared with povidone iodine, infiltrated with bupivacaine (0.25%)and a flank skin incision was made parallel to the spine at the 12thcostal level. After incision of the skin and subcutaneous thoracolumbarfascia, the longissimus lumborum and iliocostalis lumborum muscles wereretracted. The abdominal aorta was exposed via a left retroperitonealapproach and mobilized just inferior to the left renal artery. A pieceof PE-60 tubing was looped around the aorta immediately distal to theleft renal artery and both ends passed through a larger rubber tube. Bypulling on the PE tubing, the aorta was non-traumatically occluded for20 minutes. Heparin (400 IU) was administrated as an intravenous bolusbefore aortic occlusion. After 20 minutes of occlusion, the tube andcatheter were removed, the incision was closed and the animals weremonitored until full recovery and then were serially assessed forneurological function.

36 animals were randomly divided into six groups. In a control group(I), animals (n=6) received normal saline intravenously immediatelyafter release of aortic occlusion. Group (II) received rhEPO @ 6.5microgram/kg-bw; group (III) received a tissue protective cytokine(carbamylated erythropoietin) @ 6.5 microgram/kg-bw; group (IV) receivedanother tissue protective cytokine (asialoerythropoietin) @ 6.5microgram/kg-bw; group (V) received the same tissue protective cytokineas group (IV) but @ 20 microgram/kg-bw; and group (VI) received yetanother tissue protective cytokine (asialocarbamylatederythropoietin) @20 microgram/kg-bw all intravenously immediately after reperfusion (n=6for each group).

Motor function was assessed according to the criteria of Drummond andMoore by an investigator blind to the treatment at 1, 24 and 48 h afterreperfusion. A score of 0 to 4 was assigned to each animal as follows:0=paraplegic with no evident lower extremity motor function; 1=poorlower extremity motor function, weak antigravity movement only;2=moderate lower extremity function with good antigravity strength butinability to draw legs under body; 3=excellent motor function with theability to draw legs under body and hop, but not normally; 4=normalmotor function. The urinary bladder was evacuated manually in paraplegicanimals twice a day.

FIG. 22 is a graph plotting motor function of the recovering rabbits.The graph demonstrates that even over a period of only two dayserythropoietin and the tissue protective cytokines of the presentinvention permit the rabbits to recover more fully from the spinal cordinjury. Similar results would be expected from the therapeutic treatmentwith the tissue protective cytokines of the present invention.

Example 12 Anti-Inflammatory Affects of Erythropoietin

In-Vivo Studies:

1. Middle Cerebral Artery Occlusion (MCAO) Studies on Rats.

Male Crl:CD(SD)BR rats weighing 250-280 g were obtained from CharlesRiver, Calco, Italy. Surgery was performed on these rats in accordancewith the teachings of Brines, M. L., Ghezzi, P., Keenan, S., Agnello,D., de Lanerolle, N. C., Cerami, C., Itri, L. M., and Cerami, A. 2000Erythropoietin crosses the blood-brain barrier to protect againstexperimental brain injury [In Process Citation] Proc Natl Acad Sci USA97:10526-10531. Briefly, the rats were anesthetized with chloral hydrate(400 mg/kg-bw, i.p.), the carotid arteries were visualized, and theright carotid was occluded by two sutures and cut. A burr hole adjacentand rostral to the right orbit allowed visualization of the MCA, whichwas cauterized distal to the rhinal artery. To produce a penumbra(borderzone) surrounding this fixed MCA lesion, the contralateralcarotid artery was occluded for 1 hour by using traction provided by afine forceps and then re-opened. PBS or rhEPO (5,000 U/kg-bw, i.p.;previously shown to be protective in this model (1)) were administeredimmediately after the MCAO. When indicated, TNF and IL-6 were quantifiedin brain cortex homogenates as previously described (8). MCP-1 wasmeasured in the homogenates using a commercially available ELISA kit(biosource, Camarillo, Calif.).

Twenty-four hours after MCAO, the rats were anesthetized as describedabove and transcardially perfused with 100 ml saline followed by 250 mlof sodium phosphate buffered 4% paraformaldehyde solution. Brains wererapidly removed, fixed in sodium phosphate buffered 4% paraformaldehydesolution for two hours, transferred to 20% sucrose solution in PBSovernight, then in 30% sucrose solution until they sank and were thenfrozen in 2-methylbutane at −45° C. Sections (30 μm) were cut on acryostat (HM 5000M, Microm) at −20° C. in the transverse plane throughthe brain and selected every fifth section for histochemistry againstthe different antigens, or hematoxylin-eosin staining Free floatingsections were processed for immunoreactivity both with anti-glialfibrillary acid protein (GFAP) mouse monoclonal antibody (1:250,Boehringher Mannheim, Monza, Italy) and with anti-cd11b (MRC OX-42)mouse monoclonal antibody (1:50, Serotec, UK), according to theprotocols described by Houser et al. and the manufacturer's protocolrespectively. All sections were mounted for light microscopy in salineon coated slides, dehydrated through graded alcohols, fixed in xyleneand coverslipped using DPX mountant (BDH, Poole, UK). Adjacent sectionswere stained with hematoxylin-eosin as described (10).

FIG. 23 shows a coronal section of the brain cortical layer stained byhematoxilyn and eosin. Control rat (A), ischemic rat treated with PBS(B), ischemic rat treated with rhEPO (5,000 U/kg-bw, i.p., immediatelyfollowing MCAO) (C). The section B shows a marked decrease in tissuestaining consistent with inflammation, accompanied by a loss of neuronalcomponent compared to the control (A). Systemic rhEPO administrationlargely reduces the ischemic damage localizing the cell death or injuryin a restricted area (C). (Magnification 2.5×. Size bar=800 μm.)

FIG. 24 shows coronal sections of frontal cortex adjacent to the regionof infarction stained by GFAP antibody. Control rat (A), ischemic rattreated with PBS (B), ischemic rat treated with rhEPO(C). Activatedastrocytes are visualized by their GFAP-positive processes (Panel B).There was a marked reduction in number as well as staining intensity ofactivated astrocytes in a representative rhEPO-treated animal (Panel C).(Magnification 10×. Size bar=200 μm.)

FIG. 25 shows coronal sections of brain cortical layer stained by OX-42antibody. Ischemic rat treated with PBS (A), ischemic rat treated withrhEPO (B). In the ischemic cerebral hemisphere the cellular staining isespecially prominent around the infarcted tissue in both treatmentgroups, but is much denser and extends further in the saline treatedgroup. (Magnification 20×; Size bar=100 μm).

FIG. 26 shows coronal sections of brain cortical layer adjacent to theregion of infarction stained by OX-42 antibody. A much higher density ofmononuclear inflammatory cells are observed in the tissue from anischemic rat treated with PBS (A) compared to an ischemic rat treatedwith rhEPO (B). The infiltrating leukocytes, with typical round shape,potentially will extend the volume of infarction. (Magnification 10×;Size bar=200 μm)

Similar results would be expected from the therapeutic treatment withthe tissue protective cytokines of the present invention.

2. Acute Experimental Allergic Encephalomyelitis (EAE) in Lewis Rats

Female Lewis rats, 6-8 weeks of age, were purchased from Charles River(Calco, Italy). EAE was induced in rats by injecting 50 μg of guinea pigMBP (Sigma, St. Louis, Mo.) in water emulsified in equal volumes ofcomplete Freund's adjuvant (CFA, Sigma) additioned with 7 mg/ml ofheat-killed M. tuberculosis H37Ra (Difco, Detroit, Mich.) in a finalvolume of 100μ under light ether anesthesia into both hind footpads.Rats were examined in a blinded fashion for signs of EAE and scored asfollows: 0, no disease; 1, flaccid tail; 2, ataxia; 3, complete hindlimb paralysis with urinary incontinence. Starting from day 3 afterimmunization, rats were given r-Hu-EPO (EPOetin alfa, Procrit, OrthoBiotech, Raritan, N.J.) intraperitoneally (i.p.) once a day at theindicated doses, in PBS. Since the clinical-grade EPO contained humanserum albumin, control animals were always given PBS containing anidentical amount of human serum albumin. Daily administration of 5,000U/kg-bw of EPO increased the hematocrit by 30% (data not shown). Whenindicated, rats were injected s.c. once a day from day 3 until day 18with 1.3 mg/kg-bw dexamethasone (DEX) phosphate disodium salt (Sigma)corresponding to 1 mg/kg-bw of DEX, dissolved in PBS. When indicated,TNF and IL-6 were quantified in brain and spinal cord homogenates aspreviously described [Agnello, 2000 #10].

FIG. 27 shows the protective effect on the clinical signs of EAE ofdifferent doses of EPO, given from day 3 after immunization with MBPuntil day 18. EPO, in a dose-dependent fashion, delayed the onset ofdisease and decreased disease severity. But, EPO did not delay the timeto greatest severity.

In experiments where treatment of EPO was discontinued after the diseaseregressed and the rats were monitored up to two months, no relapse wasobserved, in contrast with DEX which induces an exacerbation of diseaseafter suspending its administration (FIG. 28). Similar results would beexpected from the therapeutic treatment with the tissue protectivecytokines of the present invention.

In Vitro Studies:

Primary cultures of glial cells were prepared from new bornSprague-Dawley rats 1-2 days old. Cerebral hemispheres were freed fromthe meninges and mechanically disrupted. Cells were dispersed in asolution of trypsin 2.5% and DNAase 1%, filtered through a 100 μm nylonmesh and plated (140,000 cells per 35 mm dish) in Eagle's minimumessential medium supplemented with 10% fecal calf serum, 0.6% glucose,streptomycin (0.1 mg/ml) and penicillin (100 Ul/ml). Glial cultures werefed twice a week and grown at 37° C. in a humidified incubator with 5%CO₂. All experiments were performed on 2-3 week-old glial cell cultureswith 97% astrocytes and 3% microglia, as assessed by immunochemistryoGFAP and Griffonia simplicifolia isolectin B₄. Neuronal cultures wereestablished from the hippocampus of 18-day rat fetuses. Brains wereremoved and freed from meninges and the hippocampus was isolated. Cellswere dispersed by incubation for 15-20 min at 37° C. in a 2.5% trypsinsolution followed by tituration. The cell suspension was diluted in themedium used for glial cells and plated onto polyornithine-coatedcoverslips at a density of 160,000 cells per coverslip. The day afterplating, coverslips were transferred to dishes containing a glialmonolayer in neuron maintenance medium (Dulbecco's modified Eagle'smedium and Ham's nutrient mix F12 supplemented with 5 μg/ml insulin, 100μg/ml transferrin, 100 μg/ml putrescin, 30 nM Na selenite, 20 nMprogesterone and penicillin 100 U/ml) supplemented with cytosinearabinoside 5 μM. Coverslips were inverted so that the hippocampalneurons faced the glia monolayer. Paraffin dots adhering to thecoverslips supported them above the glia, creating a narrow gap thatprevented the two cell types from contacting each other but allowed thediffusion of soluble substances. These culture conditions allowed thegrowth of differentiated neuronal cultures with >98% homogeneity, asassessed by immunochemistry of microtubule-associated protein 2 andGFAP. Cells were then treated for 24 hours with 1 μM Trimethyl tin(TMT), in the presence or absence of rhEPO (10 U (80 ng)/ml), thesupernatants used for TNF assay and cellular viability evaluated asdescribed below. When indicated, glial cells were cultured in thepresence of LPS for 24 hours, with or without rhEPO, and TNF measured inthe cultured supernatants. Cell viability was measured by the3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. Denizot, F., and Lang, R. 1986. Rapid colorimetric assay for cellgrowth and survival. Modifications to the tetrazolium dye proceduregiving improved sensitivity and reliability. J Immunol Methods89:271-277. Briefly, MTT tetrazolium salt was dissolved in serum-freemedium to a final concentration of 0.75 mg/ml and added to the cells atthe end of the treatment for 3 h at 37° C. The medium was then removedand the formazan was extracted with 1N HCl:isopropanol (1:24).Absorbance at 560 nm was read on a microplate reader.

FIG. 29 shows that rhEPO prevents neuronal death-induced TNF productionin mixed neuron-glia cultures. Panel A: Percentage of neural cell deathinduced by TMT 1 μM without or with treatment with rhEPO (10 U/ml).Panel B: Release of TNF-______ from glial cells exposed to TMT 1 μM inthe presence (hatched bars) or absence (filled bars) of neurons, with orwithout rhEPO (10 U/ml). Similar results would be expected from thetherapeutic treatment with the tissue protective cytokines of thepresent invention.

The invention is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the invention, and functionally equivalent methods andcomponents are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference herein intheir entireties for all purposes.

1.-49. (canceled)
 50. A method for treating or protecting against tissueinjury in a mammal, comprising administering to a mammal in need thereofa carbamylated erythropoietin having at least one carbamylated lysineresidue or carbamylated N-terminal amino group, wherein saidcarbamylated erythropoietin has: (a) a reduced level of in vivoerythropoietic activity compared to native erythropoietin as determinedby the exhypoxic polycythemic mouse bioassay, and (b) tissue protectiveactivity in vivo as determined by the middle cerebral artery occlusiontest, and wherein the injury is caused by stroke.
 51. The method ofclaim 50 wherein said carbamylated erythropoietin is selected from thegroup comprising alpha-N-carbamoylerythropoietin;N-epsilon-carbamoylerythropoietin; alpha-N-carbamoyl,N-epsilon-carbamoylerythropoietin;alpha-N-carbamoylasialoerythropoietin;N-epsilon-carbamoylasialoerythropoietin; alpha-N-carbamoyl,N-epsilon-carbamoylasialoerythropoietin;alpha-N-carbamoylhyposialoerythropoietin;N-epsilon-carbamoylhyposialoerythropoietin; or alpha-N-carbamoyl,N-epsilon-carbamoylhyposialoerythropoietin.
 52. A method for treating orprotecting against issue injury in a mammal, comprising administering toa mammal in need thereof a carbamylated erythropoietin having at leastone carbamylated lysine residue or carbamylated N-terminal amino group,wherein said carbamylated erythropoietin has: (a) a reduced level of invivo erythropoietic activity compared to native erythropoietin asdetermined by the exhypoxic polycythemic mouse bioassay, and (b) tissueprotective activity in vivo as determined by the middle cerebral arteryocclusion test, and wherein the injury is caused by cerebral ischemia.53. The method of claim 52 wherein said carbamylated erythropoietin isselected from the group comprising alpha-N-carbamoylerythropoietin;N-epsilon-carbamoylerythropoietin; alpha-N-carbamoyl,N-epsilon-carbamoylerythropoietin;alpha-N-carbamoylasialoerythropoietin;N-epsilon-carbamoylasialoerythropoietin; alpha-N-carbamoyl,N-epsilon-carbamoylasialoerythropoietin;alpha-N-carbamoylhyposialoerythropoietin;N-epsilon-carbamoylhyposialoerythropoietin; or alpha-N-carbamoyl,N-epsilon-carbamoylhyposialoerythropoietin.
 54. A method for treating orprotecting against tissue injury comprising administering to a mammal inneed thereof carbamylated erythropoietin having at least onecarbamylated lysine residue or carbamylated N-terminal amino group,wherein said carbamylated erythropoietin has: (a) a reduced level of invivo erythropoietic activity compared to native erythropoietin asdetermined by the exhypoxic polycythemic mouse bioassay, and (b) tissueprotective activity in vivo as determined by the middle cerebral arteryocclusion test, and wherein the injury is caused by retinal ischemia.55. The method of claim 54 wherein said carbamylated erythropoietin isselected from the group comprising alpha-N-carbamoylerythropoietin;N-epsilon-carbamoylerythropoietin; alpha-N-carbamoyl,N-epsilon-carbamoylerythropoietin;alpha-N-carbamoylasialoerythropoietin;N-epsilon-carbamoylasialoerythropoietin; alpha-N-carbamoyl,N-epsilon-carbamoylasialoerythropoietin;alpha-N-carbamoylhyposialoerythropoietin;N-epsilon-carbamoylhyposialoerythropoietin; or alpha-N-carbamoyl,N-epsilon-carbamoylhyposialoerythropoietin.